Compositions and methods for topical application and transdermal delivery of botulinum toxins

ABSTRACT

Improved formulations for transdermal delivery of  botulinum  toxin are disclosed. The formulations include, for example,  botulinum  toxin non-covalently associated with a positively charged backbone having branching or efficiency groups. The formulations also include a partitioning agent, oligo-bridge, or polyanion bridge, and may optionally contain a viscosity modifying agent. The formulations are designed for topical application onto the skin of a patient and may be used to treat wrinkles, hyperhidrosis, and other health-related problems. Kits for administration are also described.

This application claims the benefit of U.S. Provisional PatentApplication No. 60/658,434, filed on Mar. 3, 2005. U.S. PatentApplication No. 60/658,434 is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Skin protects the body's organs from external environmental threats andacts as a thermostat to maintain body temperature. It consists ofseveral different layers, each with specialized functions. The majorlayers include the epidermis, the dermis and the hypodermis. Theepidermis is a stratifying layer of epithelial cells that overlies thedermis, which consists of connective tissue. Both the epidermis and thedermis are further supported by the hypodermis, an internal layer ofadipose tissue.

The epidermis, the topmost layer of skin, is only 0.1 to 1.5 millimetersthick (Inlander, Skin, New York, N.Y.: People's Medical Society, 1-7(1998)). It consists of keratinocytes and is divided into several layersbased on their state of differentiation. The epidermis can be furtherclassified into the stratum corneum and the viable epidermis, whichconsists of the granular melphigian and basal cells. The stratum corneumis hygroscopic and requires at least 10% moisture by weight to maintainits flexibility and softness. The hygroscopicity is attributable in partto the water-holding capacity of keratin. When the horny layer loses itssoftness and flexibility it becomes rough and brittle, resulting in dryskin.

The dermis, which lies just beneath the epidermis, is 1.5 to 4millimeters thick. It is the thickest of the three layers of the skin.In addition, the dermis is also home to most of the skin's structures,including sweat and oil glands (which secrete substances throughopenings in the skin called pores, or comedos), hair follicles, nerveendings, and blood and lymph vessels (Inlander, Skin, New York, N.Y.:People's Medical Society, 1-7 (1998)). However, the main components ofthe dermis are collagen and elastin.

The hypodermis is the deepest layer of the skin. It acts both as aninsulator for body heat conservation and as a shock absorber for organprotection (Inlander, Skin, New York, N.Y.: People's Medical Society,1-7 (1998)). In addition, the hypodermis also stores fat for energyreserves. The pH of skin is normally between 5 and 6. This acidity isdue to the presence of amphoteric amino acids, lactic acid, and fattyacids from the secretions of the sebaceous glands. The term “acidmantle” refers to the presence of the water-soluble substances on mostregions of the skin. The buffering capacity of the skin is due in partto these secretions stored in the skin's horny layer.

One of the principal functions of skin is to provide a barrier to thetransportation of water and substances potentially harmful to normalhomeostasis. The body would rapidly dehydrate without a tough,semi-permeable skin. The skin helps to prevent the entry of harmfulsubstances into the body.

Wrinkles, one of the telltale signs of aging, can be caused bybiochemical, histological, and physiologic changes that accumulate fromenvironmental damage (Benedetto, International Journal of Dermatology,38:641-655 (1999)). In addition, there are other secondary factors thatcan cause characteristic folds, furrows, and creases of facial wrinkles(Stegman et al., The Skin of the Aging Face Cosmetic DermatologicalSurgery, 2^(nd) ed., St. Louis, Mo.: Mosby Year Book: 5-15 (1990)).These secondary factors include the constant pull of gravity, frequentand constant positional pressure on the skin (i.e., during sleep), andrepeated facial movements caused by the contraction of facial muscles(Stegman et al., The Skin of the Aging Face Cosmetic DermatologicalSurgery, 2^(nd) ed., St. Louis, Mo.: Mosby Year Book: 5-15 (1990)).

Different techniques have been utilized in order potentially to mollifysome of the signs of aging. These techniques range from facialmoisturizers containing alpha hydroxy acids and retinol to surgicalprocedures and injections of neurotoxins. For example, in 1986, Jean andAlastair Carruthers, a husband and wife team consisting of an ocuplasticsurgeon and a dermatologist, began to evolve the cosmetic use of thetype A form of botulinum toxin for treatment of movement-associatedwrinkles in the glabella area (Schantz and Scott, In Lewis G E (Ed)Biomedical Aspects of Botulinum, New York: Academic Press, 143-150(1981)). The Carruthers' use of botulinum type A for the treatment ofwrinkles led to their seminal publication of this approach in 1992(Schantz and Scott, In Lewis G E (Ed) Biomedical Aspects of Botulinum,New York: Academic Press, 143-150 (1981)). By 1994, the same teamreported experiences with other movement-associated wrinkles on the face(Scott, Opthalmol, 87:1044-1049 (1980)). This in turn led to the birthof the era of cosmetic botulinum type A treatment.

In addition to botulinum type A, there are seven other botulinum toxinsthat are serologically related, but distinct. Generally, botulinumtoxins (also known as botulin toxins or botulinum neurotoxins) areneurotoxins produced by the gram-positive bacteria Clostridiumbotulinum. They act to produce paralysis of muscles by preventingsynaptic transmission or release of acetylcholine across theneuromuscular junction, and are thought to act in other ways as well.Their action essentially blocks signals that normally would cause musclespasms or contractions, resulting in paralysis.

Of the eight serologically related botulinum toxins, seven can causeparalysis, namely botulinum neurotoxin serotypes A, B, C, D, E, F and G.Each of these is distinguished by neutralization with type-specificantibodies. Nonetheless, the molecular weight of the botulinum toxinprotein molecule, for all seven of these active botulinum toxinserotypes, is about 150 kD. As released by the bacterium, the botulinumtoxins are complexes comprising the 150 kD botulinum toxin proteinmolecule in question along with associated non-toxin proteins. Thebotulinum toxin type A complex can be produced by Clostridia bacteriumas 900 kD, 500 kD and 300 kD forms. Botulinum toxin types B and C areapparently produced as only a 700 kD or 500 kD complex. Botulinum toxintype D is produced as both 300 kD and 500 kD complexes. Botulinum toxintypes E and F are produced as only approximately 300 kD complexes. Thecomplexes (i.e. molecular weight greater than about 150 kD) are believedto contain a non-toxin hemaglutinin protein and a non-toxin andnon-toxic nonhemaglutinin protein. These two non-toxin proteins (whichalong with the botulinum toxin molecule comprise the relevant neurotoxincomplex) may act to provide stability against denaturation to thebotulinum toxin molecule and protection against digestive acids whentoxin is ingested. Additionally, it is possible that the larger (greaterthan about 150 kD molecular weight) botulinum toxin complexes may resultin a slower rate of diffusion of the botulinum toxin away from a site ofintramuscular injection of a botulinum toxin complex.

The different serotypes of botulinum toxin vary in the animal speciesthat they affect and in the severity and duration of the paralysis theyevoke. For example, it has been determined that botulinum toxin type Ais 500 times more potent, as measured by the rate of paralysis producedin the rat, than is botulinum toxin type B. Additionally, botulinumtoxin type B has been determined to be non-toxic in primates at a doseof 480 U/kg, about 12 times the primate LD₅₀ for type A. Due to themolecule size and molecular structure of botulinum toxin, it cannotcross stratum corneum and the multiple layers of the underlying skinarchitecture.

The toxic condition resulting from systemic botulinum toxin exposure(referred to as botulism) has existed in Europe since antiquity. In1895, Emile P. van Ermengem first isolated the anaerobic spore-formingbacillus from raw salted pork meat obtained from post-mortem tissue ofvictims who died of botulism in Belgium. Van Ermengem found the diseaseto be caused by an extracellular toxin that was produced by what hecalled Bacillus botulinus (Van Ermengem, Z Hyyg Infektionskr, 26:1-56;Rev Infect (1897)). The name was changed in 1922 to Clostridiumbotulinum. The name Clostridium was used to reflect the anaerobic natureof the microorganism and also its morphologic characteristics(Carruthers and Carruthers, Can J Opthalmol, 31:389-400 (1996)). In the1920's, a crude form of Botulinum toxin type A was isolated afteradditional outbreaks of food poisoning. Dr. Herman Sommer at theUniversity of California, San Francisco made the first attempts topurify the neurotoxin (Borodic et al., Ophthalmic Plast Recostr Surg,7:54-60 (1991)). In 1946, Dr. Edward J. Schantz and his colleaguesisolated the neurotoxin in crystalline form (Schantz et al., In: JankoviJ, Hallet M (Eds) Therapy with Botulinum Toxin, New York, N.Y.: MarcelDekker, 41-49 (1994)). By 1949, Burgen and his associates were able todemonstrate that the botulinum toxin blocks impulses across theneuromuscular junction (Burgen et al., J Physiol, 109:10-24 (1949)).Allan B. Scott first used botulinum toxin A (BTX-A) in monkeys in 1973.Scott demonstrated reversible ocular muscle paralysis lasting 3 months(Lamanna, Science, 130:763-772 (1959)). Soon afterwards, BTX-A wasreported to be a successful treatment in humans for strabismus,blepharospasm, and spasmodic torticollis (Baron et al., In: Baron E J,Peterson L R, Finegold S M (Eds), Bailey & Scotts DiagnosticMicrobiology, St. Louis, Mo.: Mosby Year Book, 504-523 (1994);Carruthers and Carruthers, Adv Dermatol, 12:325-348 (1997); Markowitz,In: Strickland G T (Eds) Hunters Tropical Medicine, 7^(th) ed.Philadelphia: W.B. Saunders, 441-444 (1991)). Botulinum toxin type A issaid to be the most lethal natural biological agent known to man. Sporesof C. botulinum are found in soil and can grow in improperly sterilizedand sealed food containers. Ingestion of the bacteria can causebotulism, which can be fatal.

At the same time, the muscle-paralyzing effects of botulinum toxin havebeen used for therapeutic effects. Controlled administration ofbotulinum toxin has been used to provide muscle paralysis to treatconditions, for example, neuromuscular disorders characterized byhyperactive skeletal muscles. Conditions that have been treated withbotulinum toxin include hemifacial spasm, adult onset spasmodictorticollis, anal fissure, blepharospasm, cerebral palsy, cervicaldystonia, migraine headaches, strabismus, temperomandibular jointdisorder, and various types of muscle cramping and spasms. More recentlythe muscle-paralyzing effects of botulinum toxin have been takenadvantage of in therapeutic and cosmetic facial applications such astreatment of wrinkles, frown lines, and other results of spasms orcontractions of facial muscles.

In view of both the toxicity of botulinum toxin, as well as itspotential for therapeutic benefits, it would be desirable to developcompositions and methods for safe application of the toxin. Topicalapplication of botulinum toxin would provide for a safer and moredesirable treatment alternative due to the painless nature ofapplication, the larger treatment surface area that can be covered, theability to formulate a pure toxin with higher specific activity, thereduced training necessary for applying the botulinum therapeutic, thesmaller doses that would be necessary to produce the desired effect, andthe lack of a requirement for large wells of toxin to reach atherapeutic clinical result. An effective means for transdermal deliveryof botulinum toxin, as well as an effective means for administeringbotulinum toxin to treat or prevent a number of conditions that does notrequire injection is thus highly desirable.

SUMMARY OF THE INVENTION

This invention relates to new compositions comprising a botulinum toxin,more specifically to such compositions that enable the transport ordelivery of a botulinum toxin through the skin or epithelium (alsoreferred to as “transdermal delivery”), and that therefore may be usedas topical applications for providing a botulinum toxin to a subject,for various therapeutic, aesthetic and/or cosmetic purposes, asdescribed herein.

One aspect of this invention is to provide a composition containing abotulinum toxin and a carrier. The carrier has a polymeric backbone withattached positively charged branching groups. The association betweenthe carrier and the botulinum toxin is non-covalent.

This invention also provides a method of administering a botulinum toxinto a subject involving topically applying to the skin or epithelium ofthe subject the botulinum toxin in conjunction with an effective amountof a carrier. The carrier has a polymeric backbone with attachedpositively charged branching groups, and associates non-covalently withthe botulinum toxin.

Another aspect of this invention is to provide formulations containing abotulinum toxin, a positively charged backbone, and at and at least onemember selected from the group consisting of a partitioning agent,oligo-bridge, and polyanion bridge, such that the botulinum toxin isnon-covalently complexed with the positively charged backbone. Thisformulation can be used to treat wrinkles by applying the formulation toan area of skin. If desired, an occlusion agent may be applied afterapplication of the formulation.

The formulations of this invention can also be used to treathyperhidrosis. Treatment methods contemplated by the invention includeapplying to an area of skin of the formulations of this invention andoptionally applying an occlusion agent afterwards.

Another aspect of this invention is to provide a kit for administrationof a botulinum toxin to a subject. The kit includes a botulinum toxinpresent in an effective amount for transdermal delivery thereof, and acarrier that has a polymeric backbone with attached positively chargedbranching groups. The association between the carrier and the botulinumtoxin is non-covalent.

Yet another aspect of this invention is to provide a kit foradministration of a botulinum toxin to a subject. The kit includes adevice for delivering the botulinum toxin to the skin and a compositioncontaining a carrier having a polymeric backbone with attachedpositively charged branching groups selected from -(gly)_(n1)-(arg)_(n2)(SEQ ID NO: 1), HIV-TAT and fragments thereof, and Antennapedia PTD, inwhich the subscript n1 is an integer of from 0 to about 20, and thesubscript n2 is independently an odd integer of from about 5 to about25.

In one aspect, this invention relates to a composition comprising abotulinum toxin (as defined herein) and a carrier comprising apositively charged “backbone” having positively charged branching or“efficiency” groups, as described herein. Most preferably the positivelycharged carrier is a long-chain positively charged polypeptide or apositively charged nonpeptidyl polymer, for example, apolyalkyleneimine. The invention further relates to a method forproducing a biologic effect such as muscle paralysis, reducinghypersecretion or sweating, treating neurologic pain or migraineheadache, reducing muscle spasms, preventing or reducing acne, orreducing or enhancing an immune response, by topically applying aneffective amount of such a composition, preferably to the skin, of asubject or patient in need of such treatment. The invention also relatesto a method for producing an aesthetic or cosmetic effect, for exampleby topical application of botulinum toxin to the face instead of byinjection into facial muscles.

This invention also provides kits for preparing or formulating acomposition that comprises the carrier and the botulinum toxin, as wellas such additional items that are needed to produce a usableformulation, or a premix that may in turn be used to produce such aformulation. Alternatively the kit comprises means for separately but inconjunction administering the botulinum toxin and the carrier to asubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the results of an experiment demonstrating efficiencyof transdermal delivery of botulinum toxin using a composition of theinvention comprising a peptide backbone.

FIG. 2 is a photograph depicting the state of the hind limbs of a mousein which the area of one limb was treated with a composition of theinvention and the area of the other was treated with another botulinumtoxin-containing composition that did not contain a carrier according tothe invention.

FIGS. 3A and 3B: photographs depicting wrinkles on subject's foreheadbefore (FIG. 3A) and after (FIG. 3B) treatment with Revance's botulinumformulation topically.

FIG. 4 shows a Mikrosil cast of the forehead (A) after topical treatmentof wrinkles with Revance's botulinum formulation and (b) beforetreatment. These Mikrosil casts, which are useful because they minimizeartifacts that can result from photographing the actual subject, clearlyshow that the untreated side has deeper wrinkles.

FIG. 5: FIG. 5 shows photographs depicting Minor's starch/iodine testperformed on subject's forehead prior to treatment.

FIGS. 6A and 6B: FIG. 6a shows photographs depicting Minor'sstarch/iodine test five days after application of Revance's botulinumformulation (A) and a control formulation (B). The control formulationcontained a positively charged polylysine backbone with a molecularweight of about 21,000 and with TAT branching groups. These pictureswere taken two minutes after application. FIG. 6b is the same as FIG. 6a, except that they were taken after four minutes had elapsed.

FIG. 7: shows the dose area used in the axillary hyperhidrosis studies.Note that the dose area extends one centimeter beyond the area of theskin covered by axillary hair.

FIGS. 8A and 8B: FIG. 8a represents the results of an experimentdemonstrating efficiency of botulinum toxin therapeutically deliveredacross intact skin as a topical agent using a short peptidyl carrier forthe treatment of axillary hyperhidrosis on human subjects. Graph depictssignificant reduction in amount of sweat (mg per 5 minutes) measuredgravimetrically 4 weeks after treatment with BOTOX® plus a shortpeptidyl carrier or carrier alone. Results are 4 week values as ratio tobaseline value for same group, with significance determined by Wilcoxonanalysis with P<0.05. N=10 patients. FIG. 8b represents the results ofan experiment demonstrating efficiency of botulinum toxintherapeutically delivered across intact skin as a topical agent using ashort peptidyl carrier for the treatment of axillary hyperhidrosis onhuman subjects. Graph depicts significant reduction in amount of sweat(mg per 5 minutes) measured gravimetrically 4 weeks after treatment withBOTOX® plus a short peptidyl carrier or carrier alone. Results aretreatment values as ratio to control value for both timepoints, withsignificance determined by Wilcoxon analysis with P<0.05. N=10 patients.

FIGS. 9A, 9B, 9C and 9D: photographs depicting Minor's starch/iodinetest before and after treatment Revance's botulinum formulationtopically for the treatment of axillary hyperhidrosis. Starch/iodinetest at Baseline vs. 2 week is shown where right axilla was treated withRevance's botulinum formulation (a and c) and left axilla was appliedwith the control (b and d) for subject #12. These photographs illustratetypical benefits observed after treatment with carrier+botox in starchiodine. Although some crossover is observed on the control side(consistent with 25% reduction in gravimetric data), significantreductions are afforded with treatment (consistent with 65% reduction ingravimetric data on treated side).

FIGS. 10(a) and 10(b): FIG. 10(a) shows muscle force generationfollowing application of a control formulation to a male CD1 mouse. FIG.10(b) shows muscle force generation following application of topical“Revance Botox® solution,” as described in Example 9.

FIGS. 11(a)-11(d); FIGS. 11(a)-11(d) show mouse foot sweat productionvisualized by iodine-starch staining (blue-black positives) 7 days aftertopical application of botulinum toxin without carrier (a and c) orbotulinum toxin with KNR (b and d) in two different animals, asdescribed in Example 7.

FIGS. 12A and 12B: Modified Franz Chamber. (a) Apparatus setup,including reservoir, circulating water bath, inline peristaltic pump,inline Franz chambers, and fraction collector. (b) Cross-section of anindividual Franz chamber, showing input and output tubing, skinmembrane, and donor compound placement.

FIGS. 13A and 13B: Increased flux using K30T vs. controls. (a) K30T vs.poly-lysine (b) K30T vs. no carrier.

FIGS. 14A, 14B, 14C, 14D, 14E and 14F: The efficiency of Revance carrierin delivering Botulinum Toxin Type A across the skin barrier in aporcine skin model was evaluated using modified Franz chamber. Increasedflux of toxin after topical application is shown (FIG. 14a-f ). Eachfigure depicts the mean and standard error percentage of topical toxindelivery across porcine skin with varying carrier:toxin mass ratios.

FIGS. 15A, 15B, 15C, 15D, 15E and 15F: Representative photomicrographsdepicting streptavidin staining (blue positive) after topicalapplication of: biotinylated botulinum toxin without carrier (controlgroup, a-c); or biotinylated botulinum toxin with K30Ts carrier(treatment group, d-f).

FIG. 16: This figure shows the percentage of muscle for reduction forNNX with K15T2 (treatment group), NNX without carrier (control group),and NNX injection.

FIG. 17: Photographs showing reduce forehead wrinkles after topicalbotulinum toxin type A. Human subject 1 is in the top row and subject 2is in the bottom row. The photographs illustrate pre-treatment(baseline) and post-treatment.

FIG. 18: Human axillary hyperhidrosis study comparing a formulationaccording to the invention with a control formulation.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides compositions and methods for delivery,particularly transdermal delivery, of a botulinum toxin by topicalapplication of an appropriate formulation.

According to the present invention, a positively charged carriermolecule having efficiency groups, as described herein, has been foundsuitable as a transport system for a botulinum toxin, enabling thattoxin to be administered transdermally to muscles and/or otherskin-associated structures. The transport occurs without covalentmodification of the botulinum toxin.

By “positively charged” is meant that the carrier has a positive chargeunder at least some solution-phase conditions, more preferably under atleast some physiologically compatible conditions More specifically,“positively charged” as used herein, means that the group in questioncontains functionalities that are charged under all pH conditions, forinstance, a quaternary amine, or contains a functionality which canacquire positive charge under certain solution-phase conditions, such aspH changes in the case of primary amines. More preferably, “positivelycharged” as used herein refers to those groups that have the behavior ofassociating with anions over physiologically compatible conditions.Polymers with a multiplicity of positively-charged moieties need not behomopolymers, as will be apparent to one skilled in the art. Otherexamples of positively charged moieties are well known in the prior artand can be employed readily, as will be apparent to those skilled in theart.

Generally, the positively-charged carrier (also referred to as a“positively charged backbone”) is typically a linear chain of atoms,either with groups in the chain carrying a positive charge atphysiological pH, or with groups carrying a positive charge attached toside chains extending from the backbone. Preferably, the positivelycharged backbone itself will not have a defined enzymatic or therapeuticbiologic activity. The linear backbone is a hydrocarbon backbone whichis, in some embodiments, interrupted by heteroatoms selected fromnitrogen, oxygen, sulfur, silicon and phosphorus. The majority ofbackbone chain atoms are usually carbon. Additionally, the backbone willoften be a polymer of repeating units (e.g., amino acids,poly(ethyleneoxy), poly(propyleneamine), polyalkyleneimine, and thelike) but can be a heteropolymer. In one group of embodiments, thepositively charged backbone is a polypropyleneamine wherein a number ofthe amine nitrogen atoms are present as ammonium groups(tetra-substituted) carrying a positive charge. In another embodiment,the positively charged backbone is a nonpeptidyl polymer, which may be ahetero- or homo-polymer such as a polyalkyleneimine, for example apolyethyleneimine or polypropyleneimine, having a molecular weight offrom about 10,000 to about 2,500,000, preferably from about 100,000 toabout 1,800,000, and most preferably from about 500,000 to about1,400,000. In another group of embodiments, the backbone has attached aplurality of side-chain moieties that include positively charged groups(e.g., ammonium groups, pyridinium groups, phosphonium groups, sulfoniumgroups, guanidinium groups, or amidinium groups). The sidechain moietiesin this group of embodiments can be placed at spacings along thebackbone that are consistent in separations or variable. Additionally,the length of the sidechains can be similar or dissimilar. For example,in one group of embodiments, the sidechains can be linear or branchedhydrocarbon chains having from one to twenty carbon atoms andterminating at the distal end (away from the backbone) in one of theabove-noted positively charged groups. In all aspects of the presentinvention, the association between the carrier and the biologicallyactive agent is by non-covalent interaction, non-limiting examples ofwhich include ionic interactions, hydrogen bonding, van der Waalsforces, or combinations thereof.

In one group of embodiments, the positively charged backbone is apolypeptide having multiple positively charged sidechain groups (e.g.,lysine, arginine, ornithine, homoarginine, and the like). Preferably,the polypeptide has a molecular weight of from about 10,000 to about1,500,000, more preferably from about 25,000 to about 1,200,000, mostpreferably from about 100,000 to about 1,000,000. One of skill in theart will appreciate that when amino acids are used in this portion ofthe invention, the sidechains can have either the D- or L-form (R or Sconfiguration) at the center of attachment. Alternatively, the backbonecan be an analog of a polypeptide such as a peptoid. See, for example,Kessler, Angew. Chem. Int. Ed. Engl. 32:543 (1993); Zuckermann et al.Chemtracts-Macromol. Chem. 4:80 (1992); and Simon et al. Proc. Nat'l.Acad. Sci. USA 89:9367 (1992)). Briefly, a peptoid is a polyglycine inwhich the sidechain is attached to the backbone nitrogen atoms ratherthan the α-carbon atoms. As above, a portion of the sidechains willtypically terminate in a positively charged group to provide apositively charged backbone component. Synthesis of peptoids isdescribed in, for example, U.S. Pat. No. 5,877,278, which is herebyincorporated by reference in its entirety. As the term is used herein,positively charged backbones that have a peptoid backbone constructionare considered “non-peptide” as they are not composed of amino acidshaving naturally occurring sidechains at the α-carbon locations.

A variety of other backbones can be used employing, for example, stericor electronic mimics of polypeptides wherein the amide linkages of thepeptide are replaced with surrogates such as ester linkages, thioamides(—CSNH—), reversed thioamide (—NHCS—), aminomethylene (—NHCH₂—) or thereversed methyleneamino (—CH₂NH—) groups, keto-methylene (—COCH₂—)groups, phosphinate (—PO₂RCH₂—), phosphonamidate and phosphonamidateester (—PO₂RNH—), reverse peptide (—NHCO—), trans-alkene (—CR═CH—),fluoroalkene (—CF═CH—), dimethylene (—CH₂CH₂—), thioether (—CH₂S—),hydroxyethylene (—CH(OH)CH₂—), methyleneoxy (—CH₂O—), tetrazole (CN₄),sulfonamido (—SO₂NH—), methylenesulfonamido (—CHRSO₂NH—), reversedsulfonamide (—NHSO₂—), and backbones with malonate and/orgem-diamino-alkyl subunits, for example, as reviewed by Fletcher et al.((1998) Chem. Rev. 98:763) and detailed by references cited therein.Many of the foregoing substitutions result in approximately isostericpolymer backbones relative to backbones formed from α-amino acids.

In each of the backbones provided above, sidechain groups can beappended that carry a positively charged group. For example, thesulfonamide-linked backbones (—SO₂NH— and —NHSO₂—) can have sidechaingroups attached to the nitrogen atoms. Similarly, the hydroxyethylene(—CH(OH)CH₂—) linkage can bear a sidechain group attached to the hydroxysubstituent. One of skill in the art can readily adapt the other linkagechemistries to provide positively charged sidechain groups usingstandard synthetic methods.

In one embodiment, the positively charged backbone is a polypeptidehaving branching groups (also referred to as efficiency groups). As usedherein, an efficiency group or branching group is any agent that has theeffect of promoting the translocation of the positively charged backbonethrough a tissue or cell membrane. Non-limiting examples of branching orefficiency groups include -(gly)_(n1)-(arg)_(n2) (SEQ ID NO: 1), HIV-TATor fragments thereof, or the protein transduction domain ofAntennapedia, or a fragment thereof, in which the subscript n1 is aninteger of from 0 to 20, more preferably 0 to 8, still more preferably 2to 5, and the subscript n2 is independently an odd integer of from about5 to about 25, more preferably about 7 to about 17, most preferablyabout 7 to about 13. Still further preferred are those embodiments inwhich the HIV-TAT fragment has the formula(gly)_(p)-RGRDDRRQRRR-(gly)_(q) (SEQ ID NO: 2),(gly)_(p)-YGRKKRRQRRR-(gly)_(q) (SEQ ID NO: 3) or(gly)_(p)-RKKRRQRRR-(gly)_(q) (SEQ ID NO: 4) wherein the subscripts pand q are each independently an integer of from 0 to 20 and the fragmentis attached to the backbone via either the C-terminus or the N-terminusof the fragment. Preferred HIV-TAT fragments are those in which thesubscripts p and q are each independently integers of from 0 to 8, morepreferably 2 to 5. In another preferred embodiment the positivelycharged side chain or branching group is the Antennapedia (Antp) proteintransduction domain (PTD), or a fragment thereof that retains activity.Preferably the positively charged carrier includes side-chain positivelycharged branching groups in an amount of at least about 0.05%, as apercentage of the total carrier weight, preferably from about 0.05 toabout 45 weight %, and most preferably from about 0.1 to about 30 weight%. For positively charged branching groups having the formula-(gly)_(n1)-(arg)_(n2) (SEQ ID NO: 1), the most preferred amount is fromabout 0.1 to about 25%.

In another embodiment, the backbone portion is a polylysine andpositively charged branching groups are attached to the lysine sidechainamino groups. The polylysine may have a molecular weight of from about10,000 to about 1,500,000, preferably from about 25,000 to about1,200,000, and most preferably from about 100,000 to about 1,000,000. Itcan be any of the commercially available (Sigma Chemical Company, St.Louis, Mo., USA) polylysines such as, for example, polylysine havingMW >70,000, polylysine having MW of 70,000 to 150,000, polylysine havingMW 150,000 to 300,000 and polylysine having MW >300,000. The selectionof an appropriate polylysine will depend on the remaining components ofthe composition and will be sufficient to provide an overall netpositive charge to the composition and provide a length that ispreferably from one to four times the combined length of the negativelycharged components. Preferred positively charged branching groups orefficiency groups include, for example,-gly-gly-gly-arg-arg-arg-arg-arg-arg-arg (-Gly₃Arg₇) (SEQ ID NO: 5) orHIV-TAT. In another preferred embodiment the positively charged backboneis a long chain polyalkyleneimine such as a polyethyleneimine, forexample, one having a molecular weight of about 1,000,000.

The positively charged backbones or carrier molecules comprisingpolypeptides or polyalkyleneimines, having the branching groupsdescribed above, are novel compounds and form an aspect of thisinvention.

In one embodiment of the invention, only a positively charged carrierthat has positively charged branching groups is necessary fortransdermal delivery of the botulinum toxin. In certain embodiments, thepositively charged carrier is a polypeptide (e.g., lysine, arginine,ornithine, homoarginine, and the like) having multiple positivelycharged side-chain groups, as described above. Preferably, thepolypeptide has a molecular weight of at least about 10,000. In anotherembodiment, the positively charged carrier is a nonpeptidyl polymer suchas a polyalkyleneimine having multiple positively charged side-chaingroups having a molecular weight of at least about 100,000. Suchpolyalkyleneimines include polyethylene- and polypropyleneimines. Ineither instance, for use as the sole necessary agent for transdermaldelivery the positively charged carrier molecule includes positivelycharged branching or efficiency groups, comprising-(gly)_(n1)-(arg)_(n2) (SEQ ID NO: 1), in which the subscript n1 is aninteger of from 0 to 20 more preferably 0 to 8, still more preferably 2to 5, and the subscript n2 is independently an odd integer of from about5 to about 25, more preferably from about 7 to about 17, and mostpreferably from about 7 to about 13, HIV-TAT or fragments thereof, orAntennapedia PTD or a fragment thereof. Preferably the side-chain orbranching groups have the general formula -(gly)_(n1)-(arg)_(n2) (SEQ IDNO: 1) as described above. Other preferred embodiments are those inwhich the branching or efficiency groups are HIV-TAT fragments that havethe formula (gly)_(p)-RGRDDRRQRRR-(gly)_(q) (SEQ ID NO: 2),(gly)_(p)-YGRKKRRQRRR-(gly)_(q) (SEQ ID NO: 3), or(gly)_(p)-RKKRRQRRR-(gly)_(q) (SEQ ID NO: 4), wherein the subscripts pand q are each independently an integer of from 0 to 20 and the fragmentis attached to the carrier molecule via either the C-terminus or theN-terminus of the fragment. The side branching groups can have eitherthe D- or L-form (R or S configuration) at the center of attachment.Preferred HIV-TAT fragments are those in which the subscripts p and qare each independently integers of from 0 to 8, more preferably 2 to 5.Other preferred embodiments are those in which the branching groups areAntennapedia PTD groups or fragments thereof that retain the group'sactivity. These are known in the art, for instance, from Console et al.,J. Biol. Chem. 278:35109 (2003). Preferably, the positively chargedcarrier includes side-chain positively charged branching groups in anamount of at least about 0.05%, as a percentage of the total carrierweight, preferably from about 0.05 to about 45 weight %, and mostpreferably from about 0.1 to about 30 weight %. For positively chargedbranching groups having the formula -(gly)_(n1)-(arg)_(n2) (SEQ ID NO:1), the most preferred amount is from about 0.1 to about 25%.

In another embodiment, the carrier is a polylysine with positivelycharged branching groups attached to the lysine side-chain amino groups.The polylysine used in this particularly embodiment can be any of thecommercially available (Sigma Chemical Company, St. Louis, Mo., USA,e.g.) polylysines such as, for example, polylysine having MW >70,000,polylysine having MW of 70,000 to 150,000, polylysine having MW 150,000to 300,000 and polylysine having MW >300,000. However, preferably thepolylysine has MW of at least about 10,000. Preferred positively chargedbranching groups or efficiency groups include, for example,-gly-gly-gly-arg-arg-arg-arg-arg-arg-arg (-Gly₃Arg₇) (SEQ ID NO: 5),HIV-TAT or fragments of it, and Antennapedia PTD or fragments thereof.

In other embodiments of this invention, the carrier is a relativelyshort polylysine or polyethyleneimine (PEI) backbone (which may belinear or branched) and which has positively charged branching groups.Such carriers are useful for minimizing uncontrolled aggregation of thebackbones and botulinum toxin in a therapeutic composition, which causesthe transport efficiency to decrease dramatically. When the carrier is arelatively short linear polylysine or PEI backbone, the backbone willhave a molecular weight of less than 75,000, more preferably less than30,000, and most preferably, less than 25,000. When the carrier is arelatively short branched polylysine or PEI backbone, however, thebackbone will have a molecular weight less than 60,000, more preferablyless than 55,000, and most preferably less than 50,000. If, however,partitioning agents as described herein are included in the composition,the molecular weight of the branched polylysine and PEI backbones may beup to 75,000, while the molecular weight of the linear polylysine andPEI backbones may be up to 150,000.

The term “botulinum toxin” as used herein is meant to refer to any ofthe known types of botulinum toxin, whether produced by the bacterium orby recombinant techniques, as well as any such types that may besubsequently discovered including engineered variants or fusionproteins. As mentioned above, at the present time, seven immunologicallydistinct botulinum neurotoxins have been characterized, namely botulinumneurotoxin serotypes A, B, C, D, E, F and G, each of which isdistinguished by neutralization with type-specific antibodies. Thebotulinum toxin serotypes are available from Sigma-Aldrich and fromMetabiologics, Inc. (Madison, Wis.), as well as from other sources. Thedifferent serotypes of botulinum toxin vary in the animal species thatthey affect and in the severity and duration of the paralysis theyevoke. At least two types of botulinum toxin, types A and B, areavailable commercially in formulations for treatment of certainconditions. Type A, for example, is contained in preparations ofAllergan having the trademark BOTOX®) and of Ipsen having the trademarkDYSPORT®, and type B is contained in preparations of Elan having thetrademark MYOBLOC®.

The botulinum toxin used in the compositions of this invention canalternatively be a botulinum toxin derivative, that is, a compound thathas botulinum toxin activity but contains one or more chemical orfunctional alterations on any part or on any chain relative to naturallyoccurring or recombinant native botulinum toxins. For instance, thebotulinum toxin may be a modified neurotoxin (e.g., a neurotoxin whichhas at least one of its amino acids deleted, modified or replaced, ascompared to a native, or a recombinantly produced neurotoxin or aderivative or fragment thereof). For instance, the botulinum toxin maybe one that has been modified in a way that, for instance, enhances itsproperties or decreases undesirable side effects, but that still retainsthe desired botulinum toxin activity. The botulinum toxin may be any ofthe botulinum toxin complexes produced by the bacterium, as describedabove. Alternatively, the botulinum toxin may be a toxin prepared usingrecombinant or synthetic chemical techniques (e.g. a recombinantpeptide, a fusion protein, or a hybrid neurotoxin, as prepared fromsubunits or domains of different botulinum toxin serotypes (see U.S.Pat. No. 6,444,209, for instance)). The botulinum toxin may also be aportion of the overall molecule that has been shown to possess thenecessary botulinum toxin activity, and in such case may be used per seor as part of a combination or conjugate molecule, for instance a fusionprotein. Additionally, the botulinum toxin may be in the form of abotulinum toxin precursor, which may itself be non-toxic, for instance anontoxic zinc protease that becomes toxic on proteolytic cleavage.

This invention also contemplates the general use of combinations andmixtures of botulinum toxins, although due to their differing nature andproperties, mixtures of botulinum toxin serotypes are not generallyadministered at this time in the health-care or cosmetic industries.

Compositions of this invention are preferably in the form of products tobe applied to the skin or epithelium of subjects or patients, i.e.humans or other mammals in need of the particular treatment. The term“in need” is meant to include both pharmaceutical or health-relatedneeds, for example, treating conditions involving undesirable facialmuscle spasms, as well as cosmetic and subjective needs, for example,altering or improving the appearance of facial tissue. In general thecompositions are prepared by mixing the botulinum toxin with thecarrier, and usually with one or more additional pharmaceuticallyacceptable carriers or excipients. In their simplest form they maycontain a simple aqueous pharmaceutically acceptable carrier or diluent,such as buffered saline. However, the compositions may contain otheringredients typical in topical pharmaceutical or cosmeceuticalcompositions, including a dermatologically or pharmaceuticallyacceptable carrier, vehicle or medium, (i.e. a carrier, vehicle ormedium that is compatible with the tissues to which they will beapplied). The term “dermatologically or pharmaceutically acceptable,” asused herein, means that the compositions or components thereof sodescribed are suitable for use in contact with these tissues or for usein patients in general without undue toxicity, incompatibility,instability, allergic response, and the like. As appropriate,compositions of the invention may comprise any ingredient conventionallyused in the fields under consideration, and particularly in cosmeticsand dermatology. The compositions also may include a quantity of a smallanion, preferably a polyvalent anion, for example, phosphate, aspartate,or citrate.

In terms of their form, compositions of this invention may includesolutions, emulsions (including microemulsions), suspensions, creams,lotions, gels, powders, or other typical solid or liquid compositionsused for application to skin and other tissues where the compositionsmay be used. Such compositions may contain, in addition to the botulinumtoxin and carrier, other ingredients typically used in such products,such as antimicrobials, moisturizers and hydration agents, penetrationagents, preservatives, emulsifiers, natural or synthetic oils, solvents,surfactants, detergents, emollients, antioxidants, fragrances, fillers,thickeners, waxes, odor absorbers, dyestuffs, coloring agents, powders,and optionally including anesthetics, anti-itch additives, botanicalextracts, conditioning agents, darkening or lightening agents, glitter,humectants, mica, minerals, polyphenols, silicones or derivativesthereof, sunblocks, vitamins, and phytomedicinals.

In particularly preferred embodiments, the compositions include gellingagents and/or viscosity-modifying agents. These agents are generallyadded to increase the viscosity of the composition, so as to make theapplication of the composition easier and more accurate. Additionally,these agents help to prevent the aqueous botulinum toxin/carriersolution from drying out, which tends to cause a decrease in theactivity of the botulinum toxin. Particularly preferred agents are thosethat are uncharged and do not interfere with the botulinum toxinactivity or the efficiency of the toxin-carrier complexes in crossingskin. The gelling agents may be certain cellulose-based gelling agents,such as hydroxypropylcellulose (HPC) for example. In some embodiments,the botulinum toxin/carrier complex is formulated in a compositionhaving 2-4% HPC. Alternatively, the viscosity of a solution containing abotulinum toxin/carrier complex may be altered by adding polyethyleneglycol (PEG). In other embodiments, the botulinum toxin/carrier solutionis combined with pre-mixed viscous agents, such as Cetaphil®moisturizer.

The compositions of this invention may optionally include partitioningagents. As used herein, a “partitioning agent” is any substance oradditive that has the property of preventing or minimizing unwanted oruncontrolled aggregation of the botulinum toxin with the carriers ofthis invention. Partitioning agents may be useful, for example, when aconcentrated botulinum toxin solution must be employed due to volumeconstraints. In these cases, the partitioning agent keeps the botulinumtoxin dispersed, thereby preventing aggregation of the toxin that wouldotherwise occur without the partitioning agent. Generally, apartitioning agent is (1) non-irritating, (2) does not destroy thebotulinum toxin, (3) does not confer any increase in permeability, (4)affords reliable and stable particle sizes, (5) is uncharged, and (6)does not interfere with complexes of the toxin and the transdermalcarrier. An example of a suitable partitioning agent is ethanol (EtOH).In preferred embodiments, the EtOH is less than 20% of the composition,and most preferably, less than 5% of the composition.

By way of example, if volume constraints require reconstituting 100 U ofbotulinum toxin in 0.5 ml of solution, rather than 2.5 ml, one typicallyobserves that the botulinum toxin will exhibit undesirable aggregation,and thus lowered activity. However, by adding 1% EtOH as a dispersingagent, fully activity is maintained even after 24 hours at thisconcentration. As another example, Botox® at 1.0 ml 0.9% NaClreconstitution has full activity, while reconstitution at 0.5 ml in 1%and 5% EtOH plus 0.9% NaCl produces solutions with full activity.

In certain embodiments of this invention, oligo- or polyanion bridgesare added to the botulinum toxin compositions to improve thecomplexation of the toxin with a positively charged backbone carrier. Asis well known in the art, botulinum toxin is actually a complex ofdifferent proteins, some of which are positively charged, and some ofwhich are negatively charged. Because the exact distribution of thecomponents of the toxin varies depending on the source of the toxin, itmay be that botulinum toxin from certain sources has a lower propensityfor complexation with the positively charged backbones described herein.However, one aspect of this invention is the discovery that by adding anoligo- or polyanion bridge to such botulinum toxins, the efficiency andefficacy of topical administration is increased dramatically. Suitableexamples of such oligo-/polyanion bridges include sodium phosphate (5%),PBS, or 5% poly-L-aspartate (e.g., with a MW of 3000).

Compositions according to this invention may be in the form ofcontrolled-release or sustained-release compositions, wherein thebotulinum toxin and the carrier are encapsulated or otherwise containedwithin a material such that they are released onto the skin in acontrolled manner over time. The botulinum toxin and carrier may becontained within matrixes, liposomes, vesicles, microcapsules,microspheres and the like, or within a solid particulate material, allof which is selected and/or constructed to provide release of thebotulinum toxin over time. The botulinum toxin and the carrier may beencapsulated together (e.g., in the same capsule) or separately (inseparate capsules).

Using the compositions described herein, botulinum toxin can bedelivered to muscles underlying the skin, or to glandular structureswithin the skin, in an effective amount to produce paralysis, producerelaxation, alleviate contractions, prevent or alleviate spasms, reduceglandular output, or other desired effects. Local delivery of thebotulinum toxin in this manner could afford dosage reductions, reducetoxicity and allow more precise dosage optimization for desired effectsrelative to injectable or implantable materials.

The compositions of the invention are applied so as to administer aneffective amount of the botulinum toxin. The term “effective amount” asused herein means an amount of a botulinum toxin as defined above thatis sufficient to produce the desired muscular paralysis or otherbiological or aesthetic effect, but that implicitly is a safe amount,i.e. one that is low enough to avoid serious side effects. Desiredeffects include the relaxation of certain muscles with the aim of, forinstance, decreasing the appearance of fine lines and/or wrinkles,especially in the face, or adjusting facial appearance in other wayssuch as widening the eyes, lifting the corners of the mouth, orsmoothing lines that fan out from the upper lip, or the general reliefof muscular tension. The last-mentioned effect, general relief ofmuscular tension, can be effected in the face or elsewhere. Thecompositions of the invention may contain an appropriate effectiveamount of the botulinum toxin for application as a single-dosetreatment, or may be more concentrated, either for dilution at the placeof administration or for use in multiple applications. Through the useof the positively charged carriers of this invention, a botulinum toxincan be administered transdermally to a subject for treating conditionssuch as undesirable facial muscle or other muscular spasms,hyperhidrosis, acne, or conditions elsewhere in the body in which reliefof muscular ache or spasms is desired. The botulinum toxin isadministered topically for transdermal delivery to muscles or to otherskin-associated structures. The administration may be made, for example,to the legs, shoulders, back (including lower back), axilla, palms,feet, neck, groin, dorsa of the hands or feet, elbows, upper arms,knees, upper legs, buttocks, torso, pelvis, or any other part of thebody where administration of the botulinum toxin is desired.

Administration of botulinum toxin formulations according to theinvention may also be carried out to treat other conditions, includingtreating of neurologic pain, prevention or reduction of migraineheadache or other headache pain, prevention or reduction of acne,prevention or reduction of dystonia or dystonic contractions (whethersubjective or clinical), prevention or reduction of symptoms associatedwith subjective or clinical hyperhidosis, reducing hypersecretion orsweating, reducing or enhancing immune response, or treatment of otherconditions for which administration of botulinum toxin by injection hasbeen suggested or performed.

Administration of botulinum toxin or other therapeutic proteinsdescribed herein may also be carried out for immunization-relatedpurposes. Surprisingly, administration of botulinum toxin describedherein may be carried out to reduce immune responses. More specifically,this invention allows a botulinum toxin to be delivered by an alteredroute of administration, thereby changing the complex antigenpresentation of the agent. In this way, the invention may be useful toreduce immune response to antigens to botulinum toxin, and to facilitaterepeat administration without immune-related reduction in activity.Alternatively, the complex can be prepared and applied topically toenhance an immune response, for example to provide immunizationsrespecting various proteins, for example, for childhood immunizationswithout injections. For use in connection with immune-related activity,an “effective amount” refers to an amount of the botulinum toxinsufficient to allow a subject to mount an immune response to thebotulinum toxin after application or a series of applications of it.

Most preferably, the compositions are administered by or under thedirection of a physician or other health care professional. They may beadministered in a single treatment or in a series of periodic treatmentsover time. For transdermal delivery of botulinum toxin for the purposesmentioned above, a composition as described above is applied topicallyto the skin at a location or locations where the effect is desired. Inembodiments were an aqueous botulinum toxin/carrier solution is applieddirectly to the skin, it is preferable to cover the treated area (e.g.,with Cetaphil® moisturizer) or occlude the treated area with a barrier(e.g., Telfa), in order to prevent the solution from drying out, whichwould lead to a decrease in toxin activity. Because of its nature, mostpreferably the amount of botulinum toxin applied should be applied withcare, at an application rate and frequency of application that willproduce the desired result without producing any adverse or undesiredresults. Accordingly, for instance, topical compositions of theinvention should be applied at a rate of from about 1 U to about 20,000U, preferably from about 1 U to about 10,000 U botulinum toxin per cm²of skin surface. Higher dosages within these ranges could preferably beemployed in conjunction with controlled release materials, for instance,or allowed a shorter dwell time on the skin prior to removal.

Proper preparation of the skin surface prior to the application of thebotulinum toxin/carrier composition is important for maintaining theefficacy of the solution. For example, the introduction of surfactantson the surface of the skin for the purpose of cleaning off surface oilson the skin prior to application is surprisingly counterproductive,because the surfactants appear to destroy the activity of the botulinumtoxin. This occurs even if the skin is subsequently washed with waterseveral times before application of the botulinum toxin/carriersolution. Even extremely gentle surfactants, such as those found in babywipes, appear to cause this phenomenon. Accordingly, in preferredmethods of administering the compositions of this invention, the skin ispre-cleaned using water alone. Washing with only water also appears toimprove the transdermal transport of the botulinum toxin moderately.

Additionally, the skin may be stripped to reduce the stratum corneumlayer prior to application of the botulinum toxin/carrier complex. Inprinciple, the process of stripping the skin should lead to enhancedefficiency of transdermal transport of botulinum toxin. However, themethod used to strip the skin is important. For example,acetone-mediated reduction of the stratum corneum layer in humans oranimals appears to reduce the activity of subsequently applied botulinumtoxin. In contrast, tape stripping (i.e., applying tape on the surfaceof the skin and then removing the tape) appears to allow deeperpenetration of the botulinum toxin and dosage reduction in both mousemodels and humans. It is presumed that abrasion of the skin surface(e.g, via the use of abrasive pads) would cause a similar effect as tapestripping.

This invention also comprises devices for transdermal transmission of acomposition that contains botulinum toxin and a carrier that has apositively charged backbone with attached branching groups as definedherein. Such devices may be as simple in construction as a skin patch,or may be more complicated devices that include means for dispensing andmonitoring the dispensing of the composition, and optionally means formonitoring the condition of the subject (e.g., monitoring the reactionof the subject to the substances being dispensed).

It should be noted that the choice of materials for the construction ofthe device is important. Preferred materials for the construction ofdelivery devices are those that do not lead to a loss of activity of thebotulinum toxin/carrier solution, either through degradation or unwantedadsorption of the botulinum toxin on a surface of the device. Suchundesired behavior has been observed, for example, when botulinumtoxin/carrier in an aqueous solution contacts polypropylene surfaces,but not when the botulinum toxin/carrier solution contacts polyvinylchloride (PVC) surfaces.

Generally, the compositions can be pre-formulated and/or pre-installedin a device or can be prepared later, for example using a kit thathouses the two ingredients (botulinum toxin and carrier) separately butprovides means for combining them at or prior to the time ofapplication. The amount of carrier molecule or the ratio of it to thebotulinum toxin will depend on which carrier is chosen for use in thecomposition in question. The appropriate amount or ratio of carriermolecule in a given case can readily be determined, for example, byconducting one or more experiments, such as those described below.

In general, the invention also comprises a method for administering abotulinum toxin to a subject or patient in need thereof. The methodincludes comprising topically administering an effective amount of thebotulinum toxin in conjunction with a carrier having a positivelycharged backbone with attached positively charged branching groups, asdescribed herein. By “in conjunction with” is meant that the twocomponents (botulinum toxin and carrier) are administered in acombination procedure, which may involve either combining them in acomposition, which is subsequently administered to the subject, oradministering them separately, but in a manner such that they acttogether to provide the requisite delivery of an effective amount of thetherapeutic protein. For example, a composition containing the carriermay first be applied to the skin of the subject, followed by applying askin patch or other device containing the botulinum toxin. The botulinumtoxin may be incorporated in dry form in a skin patch or otherdispensing device, while the positively charged carrier may be appliedto the skin surface before application of the patch so that the two acttogether, resulting in the desired transdermal delivery. Thus, the twosubstances (carrier and botulinum toxin) act in combination or perhapsinteract to form a composition or combination in situ. Accordingly, theinvention also comprises a kit that includes both a device fordispensing botulinum toxin via the skin and a liquid, gel, cream or thelike that contains the carrier or backbone, and that is suitable forapplying to the skin or epithelium of a subject. Kits for administeringthe compositions of the inventions, either under direction of a healthcare professional or by the patient or subject, may also include acustom applicator suitable for that purpose.

The compositions, kits and methods of this invention allow for thedelivery of a more pure botulinum toxin with higher specific activityand potentially improved pharmacokinetics. In addition, the carrier canact as a stabilizer, reducing the need for foreign accessory proteins(e.g., human serum albumin ranging from 400-600 mg or recombinant serumalbumin ranging from 250-500 mg) and/or polysaccharide stabilizers, andcan afford beneficial reductions in immune responses to the botulinumtoxin. In addition, the compositions are suitable for use in physiologicenvironments with pH ranging from about 4.5 to about 6.3, and may thushave such a pH. The compositions according to this invention may bestored either at room temperature or under refrigerated conditions.

The following are representative examples of the invention. Theydemonstrate delivery of functional botulinum neurotoxin complexes acrossskin without requiring covalent modification of the neurotoxin to bedelivered.

EXAMPLES Example 1 Transport of a Botulinum Toxin In Vivo Using aRevance Peptidyl Carrier

This experiment demonstrates the use of a peptidyl carrier to transporta large complex containing an intact labeled protein botulinum toxinacross intact skin after a single time administration relative tocontrols.

Backbone Selection:

The positively charged backbone was assembled by conjugating -Gly₃Arg₇(SEQ ID NO: 5) to polylysine (MW 112,000) via the carboxyl of theterminal glycine to free amines of the lysine side chains at a degree ofsaturation of 18% (i.e., 18 out of each 100 lysine residues isconjugated to a -Gly₃Arg₇ (SEQ ID NO: 5)). The modified backbone wasdesignated “KNR”. The control polycation was unmodified polylysine(designated “K”, Sigma Chemical Co., St. Louis, Mo.) of the same sizeand from the same lot.

Therapeutic Agent:

BOTOX® brand of botulinum toxin A (Allergan) was selected for thisexperiment. It has a molecular weight of approximately 150,000.

Preparation of Samples:

The botulinum toxin was reconstituted according to the manufacturer'sinstructions. An aliquot of the protein was biotinylated with acalculated 12-fold molar excess of sulfo-NHS-LC biotin (PierceChemical). The labeled product was designated “Btox-b”.

In each case, an excess of polycation was employed to assemble a finalcomplex that has an excess of positive charge as in delivery of highlynegative large nucleotide complexes. A net neutral or positive chargeprevents repulsion of the protein complex from highly negative cellsurface proteoglycans and extracellular matrix. Btox-b dose wasstandardized across all groups, as was total volume and final pH of thecomposition to be applied topically. Samples were prepared as follows:

Group labeled “JMW-7”: 2.0 units of Btox-b per aliquot (i.e. 20 U total)and peptidyl carrier KNR at a calculated MW ratio of 4:1 were mixed tohomogeneity and diluted to 200 microliters with phosphate bufferedsaline. The resulting composition was mixed to homogeneity with 1.8 mlof Cetaphil® cream and aliquoted in 200 microliter portions.

Group labeled “JMW-8”: 2.0 units of Btox-b per aliquot (i.e. 20 U total)and K at a MW ratio of 4:1 were mixed to homogeneity and diluted to 200microliters with phosphate buffered saline. The resulting compositionwas mixed to homogeneity with 1.8 ml of CETAPHIL® and aliquoted in 200microliter portions.

Animal Experiments to Determine Transdermal Delivery Efficiencies afterSingle Time Treatment with Peptidyl Carriers and Labeled Btox:

Animals were anesthetized via inhalation of isoflurane duringapplication of treatments. After being anesthetized, C57 black 6 mice(n=4 per group) underwent topical application of a metered 200microliter dose of the appropriate treatment applied to the cranialportion of dorsal back skin (selected because the mouse cannot reachthis region with mouth or limbs). Animals did not undergo depilation. At30 minutes after the initial treatment, mice were euthanized viainhalation of CO₂, and treated skin segments were harvested at fullthickness by blinded observers. Treated segments were divided into threeequal portions; the cranial portion was fixed in 10% neutral bufferedformalin for 12-16 hours then stored in 70% ethanol until paraffinembedding. The central portion was snap-frozen and employed directly forbiotin visualization by blinded observers as summarized below. Thetreated caudal segment was snap frozen for solubilization studies.

Biotin visualization was conducted as follows. Briefly, each section wasimmersed for 1 hour in NeutrAvidin® buffer solution. To visualizealkaline phosphatase activity, cross sections were washed in saline fourtimes then immersed in NBT/BCIP (Pierce Scientific) for 1 hour. Sectionswere then rinsed in saline and photographed in entirety on a Nikon E600microscope with plan-apochromat lenses.

Data Handling and Statistical Analysis:

Total positive staining was determined by blinded observer via batchimage analysis using Image Pro Plus software (Media Cybernetics, SilverSpring, Md.) and was normalized to total cross-sectional area todetermine percent positive staining for each. Mean and standard errorwere subsequently determined for each group with analysis ofsignificance at 95% confidence in one way ANOVA repeated measures usingStatview software (Abacus, Berkeley, Calif.).

Results:

The mean cross-sectional area that was positive for biotinylatedbotulinum toxin was reported as percent of total area after single-timetopical administration of Btox-b with either KNR (“EB-Btox”) or K(“nl”). The results are presented in the following Table 1 and areillustrated in FIG. 1. In FIG. 1, the area positive for label wasdetermined as percent of total area after three days of once dailytreatment with “EB-Btox” which contained Btox-b and the peptidyl carrierKNR and “nl”, which contained Btox-b with polycation K as a control.Mean and standard error are depicted for each group.

TABLE 1 Mean and standard error for labeled botulinum toxin area aspercent of total cross-section after single time topical administrationof Btox-b with KNR (JMW-7) or K (JMW-8) for 30 minutes. Group Mean Std.Error JMW-7 33 5.333334 JMW-8 8.666667 0.333334 P = 0.0001 (Significantat 99%)

Example 2 Therapeutic Efficacy of a Topical Botulinum Toxin Preparationwith a Peptidyl Carrier

Example 1 demonstrated that the peptidyl transdermal carrier allowedefficient transfer of botulinum toxin after topical administration in amurine model of intact skin. However, this experiment did not indicatewhether the complex protein botulinum toxin was released in a functionalform after translocation across skin. The following experiment was thusconstructed to evaluate whether botulinum toxin can be therapeuticallydelivered across intact skin as a topical agent using this peptidylcarrier (again, without covalent modification of the protein).

The positively charged backbone was again assembled by conjugating-Gly₃Arg₇ (SEQ ID NO: 5) to polylysine (MW 112,000) via the carboxyl ofthe terminal glycine to free amines of the lysine side chains at adegree of saturation of 18% (i.e., 18 out of each 100 lysine residues isconjugated to a -Gly₃Arg₇ (SEQ ID NO: 5)). The modified backbone wasdesignated “KNR”. Control polycation was unmodified polylysine(designated “K”, Sigma Chemical Co., St. Louis, Mo.) of the same sizeand from the same lot. The same botulinum toxin therapeutic agent wasused as in Example 1, and was prepared in the same manner. Samples wereprepared as follows:

Group labeled “JMW-9”: 2.0 units of botulinum toxin per aliquot (i.e. 60U total) and peptidyl carrier KNR at a calculated MW ratio of 4:1 weremixed to homogeneity and diluted to 600 microliters with phosphatebuffered saline. The resulting composition was mixed to homogeneity with5.4 ml of Cetaphil and aliquoted in 200 microliter portions.

Group labeled “JMW-10”: 2.0 units of botulinum toxin per aliquot (i.e.60 U total) and K at a MW ratio of 4:1 were mixed to homogeneity anddiluted to 600 microliters with phosphate buffered saline. The resultingcomposition was mixed to homogeneity with 5.4 ml of CETAPHIL® andaliquoted in 200 microliter portions.

Group labeled “JMW-11”: 2.0 units of botulinum toxin per aliquot (i.e.60 U total) without polycation was diluted to 600 microliters withphosphate buffered saline. The resulting composition was mixed tohomogeneity with 5.4 ml of CETAPHIL® and aliquoted in 200 microliterportions.

Animal Experiments to Determine Therapeutic Efficacy after Single TimeTreatment with Peptidyl Carriers and Botulinum Toxin:

Animals were anesthetized via inhalation of isoflurane duringapplication of treatments. After being anesthetized, C57 black 6 mice(n=4 per group) underwent topical application of metered 400 microliterdose of the appropriate treatment applied uniformly from the toes to themid-thigh. Both limbs were treated, and treatments were randomized toeither side. Animals did not undergo depilation. At 30 minutes after theinitial treatment, mice were evaluated for digital abduction capabilityaccording to published digital abduction scores for foot mobility afterbotulinum toxin administration [Aoki, K R. A comparison of the safetymargins of botulinum neurotoxin serotypes A, B, and F in mice. Toxicon.2001 December; 39 (12): 1815-20]. Mouse mobility was also subjectivelyassessed.

Data Handling and Statistical Analysis:

Digital abduction scores were tabulated independently by two blindedobservers. Mean and standard error were subsequently determined for eachgroup with analysis of significance at 95% confidence in one way ANOVArepeated measures using Statview software (Abacus, Berkeley, Calif.).

Results:

Mean digital abduction scores after single-time topical administrationof botulinum toxin with KNR (“JMW-9”), K (“JMW-10”) or diluent withoutpolycation (“JMW-11”), are presented in Table 2 and illustrated in therepresentative photomicrograph of FIG. 2 below. The peptidyl carrier KNRafforded statistically significant functional delivery of the botulinumtoxin across skin relative to both controls, which were comparable toone another. Additional independent repetitions (total of threeindependent experiments all with identical conclusions in statisticallysignificant paralysis from topical botulinum toxin with KNR but notcontrols) of the present experiment confirmed the present findings andrevealed no significant differences between topical botulinum toxin withor without K (i.e. both controls). Interestingly, the mice consistentlyambulated toward a paralyzed limb (which occurred in 100% of treatedanimals and 0% of controls from either control group). As shown in FIG.2, a limb treated with botulinum toxin plus the control polycationpolylysine or with botulinum toxin without polycation (“toxin alone”)can mobilize digits (as a defense mechanism when picked up), but thelimbs treated with botulinum toxin plus the peptidyl carrier KNR(Revance's botulinum formulation) could not be moved.

TABLE 2 Digital abduction scores 30 minutes after single-time topicalapplication of botulinum toxin with the peptidyl carrier KNR (“JMW-9”),with a control polycation K (“JMW-10”), or alone (“JMW-11”). Group MeanStd. Error JMW-9 3.333 0.333 JMW-10 0.333 0.333 JMW-11 0.793 0.300 P =0.0351 (Significant at 95%)Conclusions:

This experiment serves to demonstrate that the peptidyl transdermalcarrier can transport a therapeutically effective amount of botulinumtherapeutic across skin without covalent modification of thetherapeutic. The experiment also confirms that botulinum toxin does notfunction when applied topically in controls.

Example 3 Therapeutic Efficacy of a Topical Botulinum Toxin Preparationwith a Nonpeptidyl Carrier

This experiment demonstrates the performance of a non-peptidyl carrierin the invention.

Methods:

Backbone Selection:

The positively charged backbone was assembled by conjugating -Gly₃Arg₇(SEQ ID NO: 5) to polyethyleneimine (PEI) MW 1,000,000 via the carboxylof the terminal glycine to free amines of the PEI side chains at adegree of saturation of 30% (i.e., 30 out of each 100 lysine residues isconjugated to a -Gly₃Arg₇ (SEQ ID NO: 5). The modified backbone wasdesignated “PEIR” to denote the large nonpeptidyl carrier. Controlpolycation was unmodified PEI (designated “PEI”, Sigma Chemical Co., St.Louis, Mo.) of the same size and from the same lot. The same botulinumtoxin therapeutic agent was used as in Example 1.

Botulinum toxin was reconstituted from the Botox product according tothe manufacturer's instructions. In each case, an excess of polycationwas employed to assemble a final complex that had an excess of positivecharge as in delivery of highly negative large nucleotide complexes. Anet neutral or positive charge prevents repulsion of the protein complexfrom highly negative cell surface proteoglycans and extracellularmatrix. The botulinum toxin dose was standardized across all groups aswas total volume and final pH of the composition to be appliedtopically. Samples were prepared as follows:

Group labeled “AZ”: 2.0 units of botulinum toxin per aliquot (i.e. 60 Utotal) and the nonpeptidyl carrier PEIR in ultrapure form at acalculated MW ratio of 5:1 were mixed to homogeneity and diluted to 600microliters with phosphate buffered saline. The resulting compositionwas mixed to homogeneity with 5.4 ml of CETAPHIL® and aliquoted in 200microliter portions.

Group labeled “BA”: 2.0 units of botulinum toxin per aliquot (i.e. 60 Utotal) and PEI at a charge ratio of 5:1 were mixed to homogeneity anddiluted to 600 microliters with phosphate buffered saline. The resultingcomposition was mixed to homogeneity with 5.4 ml of CETAPHIL® andaliquoted in 200 microliter portions.

Animal Experiments to Determine Therapeutic Efficacy after Single TimeTreatment:

Animals were anesthetized via inhalation of isoflurane duringapplication of treatments. After being anesthetized, C57 black 6 mice(n=3 per group) underwent topical application of metered 400 microliterdose of the appropriate treatment applied uniformly from the toes to themid-thigh. Both limbs were treated, and treatments were randomized toeither side. Animals did not undergo depilation. At 30 minutes after theinitial treatment, mice were evaluated for digital abduction capabilityaccording to published digital abduction scores for foot mobility afterbotulinum toxin administration [Aoki, K R. A comparison of the safetymargins of botulinum neurotoxin serotypes A, B, and F in mice. Toxicon.2001 December; 39 (12): 1815-20]. Mouse mobility was also subjectivelyassessed.

Data Handling and Statistical Analysis:

Digital abduction scores were tabulated independently by two blindedobservers. Mean and standard error were subsequently determined for eachgroup with analysis of significance at 95% confidence in one way ANOVArepeated measures using Statview software (Abacus, Berkeley, Calif.).

Results:

Mean digital abduction scores after single-time topical administrationof botulinum toxin with ultrapure PEIR (“AZ”), or control polycation PEI(“BA”), are presented in Table 3 and repetition presented as Table 4(single independent repetition for this experiment). The nonpeptidylcarrier PEIR afforded statistically significant functional delivery ofbotulinum toxin across skin relative to controls. As before, animalswere observed to walk in circles toward the paralyzed limbs.

TABLE 3 Digital abduction scores 30 minutes after single-time topicaladministration of Botox with ultrapure PEIR (“AZ”), or controlpolycation PEI (“BA”). Mean and standard error are presented. Group MeanStd. Error BA 0.833 0.307 AZ 3.917 0.083 P = 0.0002 (Significant at 99%)

TABLE 4 Digital abduction scores 40 minutes after single-time topicaladministration of Botox with ultrapure PEIR (“AZ1”), or controlpolycation PEI (“BA1”). Mean and standard error are presented. GroupMean Std. Error BA1 0.333 0.211 AZ1 3.833 0.167 P = 0.0001 (Significantat 99%)Conclusions:

This experiment demonstrated that the nonpeptidyl transdermal carriercan transport therapeutic doses of botulinum toxin across skin withoutprior covalent modification of the botulinum toxin. These findingscomplement those with peptidyl transfer agents. The option of using anonpeptidyl or a peptidyl carrier to achieve the therapeutic effect willallow tailoring to specific circumstances, environments, and methods ofapplication and add to the breadth of the transdermal delivery platformof this invention.

Example 4 Therapeutic Efficacy of a Topical Botulinum Toxin Preparationwith Peptidyl Carrier for Forehead Hyperhidrosis and Wrinkles

This experiment demonstrates that botulinum toxin can be therapeuticallydelivered across intact skin as a topical agent using this peptidylcarrier for the treatment of forehead hyperhidrosis and wrinkles onhuman subjects.

Experimental Procedure for Forehead Hyperhidrosis and Wrinkles Study:

Baseline and post-treatment photographs of the subject's forehead weretaken on a blue background using a Nikon D70 camera with Nikon TTLMacro-speedlight SB29s flash (Nikon, Inc. USA).

Baseline and post-treatment videos of the subject's forehead were takenon a blue background using a Sony Digital Handycam camcorder.

Minor's starch/iodine test was performed to visualize sweat productionusing 10% topical povidone iodine solution (Walgreen Co., Deerfield,Ill.) and Kingsford's 100% corn starch (ACH Food Companies, Inc.,Memphis, Tenn.). The subject's forehead was painted with an iodinesolution using sterile cotton balls (Johnson & Johnson Consumer ProductCompany, Skillman, N.J.), and then allowed to dry completely. The areawas lightly dusted with starch powder using sterile cotton balls. Thesweat was induced with physical activity at ambient room temperature.Dark blue-black spots appeared as the sweat dissolved the iodine andreacted with starch powder. Baseline and post-treatment photographs ofiodine-starch test were taken on a blue background using a Nikon D70camera with Nikon TTL Macro-speedlight SB29s flash. The subject'sforehead was cleansed with 70% EtOH and then deionized water.

The subject's predefined treatment area on the forehead was prepared bynon-invasive tape stripping method for stratum corneum prior totreatment application. Precut tape was applied to the treatment areawith firm pressure for few seconds. It was removed rapidly by pulling onone corner of the tape. The second tape was carefully applied to thesame area immediately after the first tape was removed. Tape-strippingwas repeated 3-5 times.

Treatment Preparation:

The BOTOX® reconstituting solution of sterile 0.9% sodium chloride(Abbott Laboratories, North Chicago, Ill.) plus 5% EtOH plus 5% shortchained polyaspartate solution labeled A-3C (Donlar BioPolymer, Inc.Bedford Park, Ill.) was prepared (i.e., for every 1.0 millilitersolution, 900 microliters of sterile 0.9% sodium chloride plus 50microliters of 100% EtOH plus 50 microliters of short chainedpolyaspartate solution). Kn21T was prepared at 1 milligram/milliliterconcentration with 0.9% sodium chloride plus 5% EtOH (i.e., 500microliters of Kn21T was aliquoted and 25 microliters of 100% EtOH wasadded). As used herein, Kn21T refers to a positively charged polylysinebackbone having a molecular weight of 21,000 and TAT branching groups.100 units of BOTOX® (Allergan, Irvine, Calif.) was reconstituted with1.0 milliliters of reconstituting solution using sterile 3 ml latex freesyringe with 18_(G)1½ (Becton Dickinson & Co., Franklin Lakes, N.J.).The reconstituted BOTOX® was carefully mixed by inversion 8 times. 200units of BOTOX® were used for each subject. Revance's botulinumformulation was prepared with 200 units of BOTOX® and Kn21T plus 5% EtOH(ie. 2.0 milliliters of BOTOX® was added to 500 microliters of Kn21Tplus 25 microliters of 100% EtOH) and sat at room temperature for 5minutes for the complexes to form.

The control solution was prepared with reconstituting solution and Kn21Tplus 5% EtOH (ie. 2.0 ml of reconstituting solution was added to 500microliters of Kn21T plus 25 microliters of 100% EtOH) and kept at roomtemperature.

Treatment Application:

The subject reclined on a table with protective covering around theeyes, face, and upper body. The treatment was applied evenly to thesubject's forehead using a pipette and massaged into the skin incircular motion with fingers while wearing powder-free, nitrile gloves.The treatment area was covered with a thin layer of CETAPHIL®moisturizing cream (Galderma, Fort Worth, Tex.) and incubated for 60minutes. After 60 minute incubation, the treatment was removed withsterile gauze pads. The gauze pads and gloves were discarded in abiohazard bag.

Results:

FIG. 3 depicts significant reduction in wrinkle length depth and widthafter topical treatment with Peptidyl carrier and botulinum combination.This experiment confirms that topically applied botulinum toxin, whencombined with transdermal carrier, can afford significant muscularparalysis to afford a cosmetic effect. FIG. 4 is a MIKROSIL® cast of thetreated skin (A) versus untreated skin (B). Wrinkles are visible on thecast of the untreated skin.

FIGS. 5 and 6 show the results of the Minor's starch/iodine test. FIG. 5shows photos taken two minutes after application, with panel (A)corresponding to the side treated with Revance's botulinum formulationand panel (B) corresponding to the side treated with a controlformulation containing Kn21T carrier alone. FIG. 6 is the same as FIG.5, except that it was taken at four minutes. Note the more pronouncedcoloration on the control formulation side, indicating that the skin onthat side is secreting more sweat. Also note that the sweating startsearlier on the untreated side.

Conclusions:

This example demonstrates that topically applied complexes of botulinumtoxin can afford significant aesthetic benefit in reducing fine andcoarse wrinkles. This transepithelial effect further confirms thatmuscle paralysis can be accomplished with appropriate carriers aftertopical application of botulinum toxin complexes such as those disclosedherein. This example thus indicates that topical application ofbotulinum toxin can lead to relief from muscle spasms such asblepharospasm or torticollis as well as relief of muscle spasm-relatedpain such as lower back pain.

Example 5 Therapeutic Efficacy of a Topical Botulinum Toxin Preparationwith Peptidyl Carrier for Axillary Hyperhidrosis

This experiment demonstrates whether botulinum toxin can betherapeutically delivered across intact skin as a topical agent usingthis peptidyl carrier for the treatment of axillary hyperhidrosis onhuman subjects (n=10 axillae per group with one axilla treated and onecontrol per patient in a randomized double-blind fashion).

Inclusion Criteria for Axillary Hyperhidrosis Study:

-   -   Age: 18 years or older    -   Healthy volunteers    -   Informed consent given and signed by the volunteer    -   Subject willing to follow instruction and return for follow-up        visits.    -   Subject has presence of pre-existing, subjective hyperhidrosis    -   Subject is NOT pregnant or planning on becoming pregnant within        the next 3 months    -   Subject lives and/or work in San Francisco or near study area    -   Subject has NOT had treatment for underarm sweating within the        past 6 months    -   Subject is NOT planning on having treatment for underarm        sweating within the next 3 months        Gravimetric Measurement Procedures:

As a part of the gravimetric measurement procedure, the subjects werefirst acclimated to the testing area. Specifically, each subject sat for15 minutes at a room temperature of 72-77° F. in the resting position.

Axillae Preparation:

(Powder-free, nitrile gloves were worn for the following procedures).The subject changed into disposable cape and bra (if a woman) or tookoff all upper body garments (if a man) so as to expose both of theaxillae fully. The dose area was predetermined to be the area covered byhair bearing skin, plus an area extending 1 cm beyond the hair bearingskin at each axilla. The dose area was cleaned with a pre-wet sterilegauze pad from a 50 ml conical tube by wiping with 5 long strokes fromtop to bottom in the same direction using one side of the gauze. Thisstep was repeated three more times with a clean pre-wet gauze pad eachtime while being careful not to irritate or abrade the skin. The gauzepads were discarded in the trash. The same wash procedure was repeatedfor the other axilla. The axilla was dried with dry sterile gauze byusing firm padding motion from top to bottom of the axilla while beingcareful not to irritate or abrade the skin. Then, the axilla was furtherdried by placing a filter paper under the axillary crease and allowingthe filter to dwell in the test site for 5 minutes following theprocedure for gravimetric assessment. The patient sat with their armsagainst his/her body in a resting position. The filter papers werediscarded in the trash. The subject was allowed to rest for 1 minutewithout axilla manipulation prior to the first gravimetric assessment.

Sweat Production Measurement (Gravimetric Measurement):

(A new pair of powder-free, nitrile gloves was donned prior to thesemeasurements). The subject held his or her hands together at the back ofhead to expose axillae fully, while being partially reclined (about 45degrees). A pre-weighed filter paper was removed from a conical storagetube and placed under the subject's axilla with the tip of the filteraligning with the center of axillary crease line. The filter paper heldin place by using fingers while the subject relaxed arms to the side ofthe body. The subject sat with both arms held tightly against his/hertrunk for five minutes. The timing started when the filter papers weresecurely placed under both of the axillae. Both axillae were measuredsimultaneously. After 5 minutes, the filter papers were removed from theaxillae and placed back into the same respective conical tubes. Thefilter paper placed first was removed first. The caps of the conicaltubes were screwed tightly to prevent the evaporation of the sweat fromthe tube. The sweat production was repeated two more times at one-minuteintervals.

Minor's Starch/Iodine Test:

The subject held his/her hands together at the back of head to exposethe axillae fully. The iodine solution was painted onto the axilla areapredetermined as before with a sterile gauze pad and allowed to air-dry.When the iodine had completely dried, a thin layer of starch was paddedonto the area covered by iodine with cotton balls. The iodine wasallowed to air-dry before the application of starch in order to reducefalse positive and background. The subject then sat with both arms heldtightly against his/her trunk. After 5 minutes, the subject raisedhis/her arms and held hands together at the back of head to exposeaxillae fully. Photographs of each axilla with left and right axilla andthe date clearly labeled were taken. The axillae were cleaned with 70%EtOH and then with sterile deionized water.

Treatment Preparation:

Kn21pr was prepared at 1 milligram/milliliter concentration with salineplus 5% EtOH (i.e., 500 microliters of Kn21pr was aliquoted and 25microliters of 100% EtOH was added). As used herein, Kn21pr refers to apositively charged polylysine backbone with a molecular weight of 21,000and branching groups comprising protected oligoarginine. 100 units ofBotox® (Allergan, Irvine, Calif.) was reconstituted with 0.75milliliters of 0.9% sodium chloride (Abbott Laboratories, North Chicago,Ill.) using sterile 3 ml latex free syringe with 18_(G)1½ (BectonDickinson and Company, Franklin Lakes, N.J.). The reconstituted Botox®was carefully mixed by inversion 8 times. 200 units of Botox® were usedfor each subject. The treatment solution was prepared with 200 units ofBotox® and Kn21pr plus 5% EtOH (i.e., 1.5 milliliters of Botox® wasadded to 500 microliters of Kn21pr plus 25 microliters of 100% EtOH) andkept at room temperature for 5 minutes to allow the complexes to form.After a 5-minute incubation period, approximately 1.0 milliliters of 4%HPC (hydroxypropylcellulose) (with 1% EtOH) was added and mixed gentlyand thoroughly with a small metal spatula. The homogenous treatmentsolution was transferred into a 3 ml syringe and syringe tip cap.(Becton Dickinson and Company, Franklin Lakes, N.J.).

The control solution was prepared with 0.9% sodium chloride and Kn21prplus 5% EtOH (ie. 1.5 milliliters of 0.9% sodium chloride was added to500 microliters of Kn21pr plus 25 microliters of 100% EtOH) and sat atroom temperature for five minutes. After incubation, approximately 1.0milliliters of 4% HPC (with 1% EtOH) was added and mixed gently andthoroughly with a small metal spatula. The homogenous control solutionwas transferred into a 3 ml syringe and syringe tip cap.

Treatment Application (Wear Powder-Free, Nitrile Gloves):

The subject held his/her hands together with interlocking fingers andplaced them on the back of the head to fully exposure the subject'saxillae. Then, the subject reclined in a chair to an angle of about 45degrees. As shown in FIG. 7, the dose area was visually mapped out (i.e.1 cm beyond the hair bearing skin) for application. The dose areas werechecked for dryness. The syringe tip cap was removed from the labeledsyringe marked “L” for left and “R” for right, and prepared forapplication onto the subject's axillae. The treatment solution wasspread evenly around the dose area with a syringe and massaged into theskin with fingers for 1 minute. The subject then placed his/her armsdown along the side of the body and incubated for 60 minutes. After 60minute incubation, the treatment was cleaned with sterile gauze pads.The gauze pads and gloves were discarded in a bio-hazard bag. Thesubject was discharged.

Results:

Revance's topical botulinum formulation reduced sweating by 65%(P=0.0137). FIG. 8a shows a comparison of sweat production at 4 weeksafter treatment (randomized by side) with Kn21pr backbone alone(control) or kn21pr backbone plus 200 U BOTOX® (ratio to baseline).Statistical analyses were performed by Wilcoxon signed ranks using NPSSwith P as noted and significance at P<0.05. [n=10 subjects].

Second Comparison:

FIG. 8b shows the ratio of treatment to control compared at baseline andat 4 weeks. The figure shows sweat production (mg per 5 minutes) 4 weeksafter axillary treatment (randomized by side) with kn21pr backbone alone(control) or kn21pr backbone plus 200 U BOTOX® (ratio of treatment tocontrol). Statistical analyses were performed by Wilcoxon signed ranksusing NPSS with P as noted and significance at P<0.05. (P=0.0195) (Pr Tp=0.0217) [n=10].

FIG. 9 shows photographs depicting Minor's starch/iodine test before andafter treatment with Revance's botulinum formulation topically for thetreatment of axillary hyperhidrosis. Starch/iodine test at baseline vs.2 weeks is shown where right axilla was treated with Revance's botulinumformulation (a and c) and left axilla was applied with the control (band d) for subject #12. These photographs illustrate typical benefitsobserved after treatment with carrier+botox in starch iodine. Althoughsome crossover is observed on the control side (consistent with 25%reduction in gravimetric data), significant reductions are afforded withtreatment (consistent with 65% reduction in gravimetric data on treatedside).

Conclusions:

This example confirms that topical application of botulinum complexesformed according to the invention disclosed herein readily affordtherapeutic benefit in reduction of sweating in a cohort of patientswith hyperhidrosis—subjective or quantitative. This effect in reducingsweating has also been afforded in the forehead case presented above andin palmar/plantar application when combined with a glove to limit spreadof formulation during dwell time. This transepithelial delivery ofbotulinum toxin complexes for therapeutic benefit confirms further thatthe approach can be extended to other cases where SNAP function oracetylcholine signals are crucial such as bladder dysfunction or spasm,gastrointestinal applications, or sebaceous gland secretions for smellreduction or acne prevention/treatment.

Example 6 Wipe-On Revance's Botulinum Formulation Pilot Experiment

Purpose:

To determine transport efficiency of wipe-on Dysport and performance ofpeptidyl transdermal carriers (backbones) in a murine model.

Methods:

Study design: C57BL/6, female mice (Charles River, Wilmington, Mass.)weighing 19-20 g were used. Animals were anesthetized using isofluraneand topical application of “Revance Dysport solutions” (Table 5) wasperformed on mouse hind limbs. After recovery, hind limb muscleweakening was scored using Digit Abduction Score (DAS) values.

TABLE 5 Description of test compounds and peptidyl transdermal carrier(backbones). Group Test Compound Backbone CL 30U DYSPORT^(®)(Formulation 1) Kn21T CM 30U DYSPORT^(®) (Formulation 1) Kn21Pr CN 30UDYSPORT^(®) (Formulation 1) KnR CO 30U DYSPORT^(®) (Formulation 2) Kn21TCP 30U DYSPORT^(®) (Formulation 2) Kn21Pr Control Saline N/A

Test Compound Preparation:

The DYSPORT® reconstituting solution of sterile 0.9% sodium chloride(Abbott Laboratories, North Chicago, Ill.) was prepared. Backbones wereprepared at 1 milligram/milliliter concentration with 0.9% sodiumchloride. 500 units of DYSPORT® (Ipsen) was reconstituted with 2.5milliliters of reconstituting solution using sterile 3 ml latex freesyringe with 18_(G)1½ (Becton Dickinson & Co., Franklin Lakes, N.J.).The reconstituted DYSPORT® was carefully mixed by inversion eight times.The Revance's botulinum formulation was prepared with 30 units ofDYSPORT® and backbone (i.e. 150 microliters of DYSPORT® was added to 75microliters of Revance carrier) in a microcentrifuge tube and sat atroom temperature for 5 minutes for the complexes to form.

Topical Application:

Animals were anesthetized using 1.5% isoflurane mixed with oxygen andthen injected with 0.05 ml rodent anesthetic cocktail (3.75 ml of 100mg/ml Ketamine, 3.00 ml of 20 mg/ml Xylazine, and 23.25 ml of saline)intraperitoneally. After being anesthetized, C57BL/6 female mice (n=3per group) were randomly divided and prepared for treatment. The animalsunderwent an acetone-strip three times. The “Revance Dysport solution”was applied to the hind limb using a pipet and massaged into the skinwearing nitrile gloves. Animals recovered in a controlled heatenvironment to prevent hypothermia. Baseline and post-treatmentphotographs, video of the animal's recovery and Digital Abduction Score(DAS) values were recorded.

Statistical Analysis:

Statistical analysis was subsequently determined for each group usingStatview® software (Abacus Concepts, Berkeley, Calif.) and expressed asmean and standard error. Statistical significance for all comparison wasdetermined using one-factor ANOVA repeated measures and Fisher PLSDpost-hoc testing at 95% confidence.

Results:

Foot Mobility Scores were tabulated using DAS values where score of 0indicates normal digit abduction (no muscle weakening) and a score of 4indicates maximal reduction in digit abduction (maximal muscleweakening) [Aoki, K. R. A Comparison of the Safety Margins of BotulinumNeurotoxin Serotypes A, B, and F in mice. Toxicon. 2001; 39:1815-1820].

Statistical analyses were determined by three different comparisons andthe results are presented in Tables 6-8. Mean DAS values showedstatistically significant muscle weakening/paralysis between treatmentgroups versus control after single-time topical administration of“Revance DYSPORT® solution.” Table 6 shows the statistically significantparalysis from topical DYSPORT® for each group (P=0.0001) versus controlwhereas Table 7 details statistically significant paralysis for alltreatment groups versus control (P=0.0013). Table 8 detailsstatistically significant paralysis for groups treated with formulation1 or 2 (P=0.0024) between groups and versus control.

After recovery, animals were observed to walk in circles toward theparalyzed limbs.

TABLE 6 Foot mobility score - DAS values. Mean and standard errors foreach group are presented after 30 minutes post treatment. Group MeanStd. Error CL 2.500 0.267 CM 1.000 0.189 CN 0.375 0.183 CO 0.250 0.164CP 0.500 0.189 Control 0.333 0.333 P = 0.0001 (Significant at 95%)

TABLE 7 Foot mobility score - DAS values. Mean and standard errors forall treatment groups versus control are presented. Group Mean Std. ErrorCL-CP 0.900 0.240 Control 0.000 0.000 P = 0.0013 (Significant at 95%)

TABLE 8 Foot mobility score - DAS values. Mean and standard errors fortreatment group with formulation 1 or 2 versus control are presented.Group Mean Std. Error CL-CN 1.400 0.400 CO, CP 0.400 0.163 Control 0.0000.000 P = 0.0024 (Significant at 95%)Conclusion:

This experiment serves to demonstrate that the peptidyl transdermalcarrier can transport a therapeutically effective amount of DYSPORT®botulinum therapeutic across skin without covalent modification of thetherapeutic.

Example 7 Sweat Inhibition by Topical Botulinum Toxin in a Mouse HindFoot Model

Purpose:

To determine the sweat inhibition by topical application of botulinumtoxin with peptidyl carrier (Revance's botulinum formulation) in amurine model.

Methods:

Study Design:

Female C57BL/6 mice (Charles River, Wilmington, Mass.) weighing 19-21 gwere used. Animals were anesthetized using 1.5% isoflurane mixed withoxygen and remained anesthetized for the duration of the study. Botoxwas applied topically at a dosage of 2 units per mouse foot (Table 9).Sweat was induced by the cholinergic drug pilocarpine. [Kaszynski, E andFrisch S B. Mouse Foot Screen for the Inhibition of Sweating byAnticholinergic Drugs. (1974) Journal of Investigative Dermatology,62:510-513]. Sweat was visualized with Minor's starch-iodine test.

TABLE 9 Description of test compounds and peptidyl transdermal carrier(backbones). Group Test Compound Backbone BO 2U BOTOX^(®) N/A BP 2UBOTOX^(®) KNR BQ 2UBOTOX^(®) KNT

Test Compound Preparation:

The pilocarpine solution (Sigma Aldrich, Cat No. P0472) was prepared 24hours prior to injection. Pilocarpine solution was prepared at 1milligram/milliliter concentration with 0.9% sodium chloride and mixedwell by vortex for 2 minutes. The pilocarpine solution was sterilized byfiltration with PURADISC 25 TF disposable filter device (Whatman, 25 mmDia. Catalog No. 6784-2504) and a large syringe into a sterile vial.Then, the solution was covered with foil. A 2% iodine solution (SigmaAldrich, Cat No. 266426) in 70% ethanol was prepared and mixed well byvortex. The iodine solution was then sonicated for 15 minutes thenvortex again. Backbones were prepared at 1 milligram/milliliterconcentration with deionized water. 100 units of BOTOX® (Allergan,Irvine, Calif.) was reconstituted with 1.0 milliliters of 0.9% sodiumchloride using sterile 3 ml latex free syringe with 18_(G)1½ (BectonDickinson & Co., Franklin Lakes, N.J.). The reconstituted BOTOX® wascarefully mixed by inversion eight times. Treatment solution wasprepared with 7 units of BOTOX® and backbone (i.e. 70 microliters ofBOTOX® was added to 35 microliters of KNR or KNT and diluted with 35microliters of PBS) in a microcentrifuge tube and sat at roomtemperature for 5 minutes for the complexes to form. Control solutionwas prepared with BOTOX® and PBS (i.e. 70 microliters of BOTOX® wasadded to 70 microliters of PBS).

Topical Application:

Animals were anesthetized using 1.5% isoflurane mixed with oxygen andthen injected with 0.07 ml rodent anesthetic cocktail (3.75 ml of 100mg/ml Ketamine, 3.00 ml of 20 mg/ml Xylazine, and 23.25 ml of saline)intraperitoneally and supplemented with isoflurane as necessary. Afterbeing anesthetized, C57BL/6 female mice (n=6 per group) were randomizedto test groups. Twenty microliters of treatment or control solution wereapplied to assigned hind feet. The bottoms of the feet were coatedcompletely and liberally with the solution. A pipette tip was used toapply a thin evenly coating of test solution to the feet. Animalsrecovered in a controlled heat environment to prevent hypothermia. Thesolutions were dried completely using heat lamp for two minutes, thenair dried for five minutes. The hind feet were then coated withapproximately 50 microliters of Cetaphil cream.

Starch-Iodine Test:

Minors starch iodine test was performed to visualize sweat distributionat baseline and at one week post treatment. Animals were kept fullyanesthetized with stable vitals for 10 minutes before the iodinesolution was applied by dipping the hind feet into 2% iodine solution.The iodine solution was dried completely using a heat lamp for threeminutes, then air dried for five minutes. Starch powder was subsequentlyapplied rubbed in with fingers while wearing powder-free gloves. Theexcess starch powder was removed with a small paint brush and then thestarch power was loosely applied with a compact velour pad to enhanceuniformity. Baseline and post-treatment photographs were recorded at 10,20 and 60 minutes post pilocarpine injection.

Results:

Animal's foot sweat was visualized by Minor's starch-iodine test. Sweatwas indicated by blue-black coloration. [Kuttner, C et al. Treatment ofGustatory Sweating with Botulinum toxin A. (2001) Int Poster J Dent OralMed. 3; 3: poster 82].

The blue-black positive spots were typically best viewed at 50-60minutes after pilocarpine injection. The starch-iodine test showed thatthe treatment groups had markedly less blue-black positive spots thanthe control as depicted in the representative photographs in FIGS.11(a)-11(d).

Example 8 Evaluating Muscle Force Generation after Topical Applicationof Revance's Botulinum Formulation

Purpose:

To evaluate the effects of neuromuscular blockade after topicalapplication of botulinum toxin type B by muscle contraction forcegeneration in a murine model.

Methods:

Study Design:

Male CD1 mice (Charles River, Wilmington, Mass.) weighing 27-33 g wereused. Mice were housed in groups of 5 and allowed ad libitum access tofood and water before treatment. Animals were anesthetized using 1.5%isoflurane mixed with oxygen and remained anesthetized for the durationof the study. A dose site of each mouse's hind limb was carefully shavedwith an Andis Edjer II cordless rechargeable trimmer (Andis, Sturtevant,Wis.). DYSPORT® was applied topically at a dosage of 25 units per mouselimb. Untreated normals, as well as those treated with base formulations(no toxin) applied topically at an equivalent volume served as controls.Muscle contraction force was measured at 2-3.5 hours post topicaltreatment.

Test Compound Preparation:

The DYSPORT® reconstituting solution of sterile 0.9% sodium chloride(Abbott Laboratories, North Chicago, Ill.) with 5% EtOH and 5%polyaspartate solution was prepared. Backbones were prepared at 1milligram/milliliter concentration with 0.9% sodium chloride. 500 unitsof DYSPORT® (Ipsen) was reconstituted with 2.5 milliliters ofreconstituting solution using sterile 3 ml latex free syringe with18_(G)1½ (Becton Dickinson & Co., Franklin Lakes, N.J.). Thereconstituted DYSPORT® was carefully mixed by inversion eight times. TheRevance's botulinum formulation was prepared with 25 units of DYSPORT®and backbone (i.e. 125 microliters of DYSPORT® was added to 62.5microliters of proprietary short peptidyl backbone based on priorexperiments above) in a microcentrifuge tube and sat at room temperaturefor 5 minutes for the complexes to form (n=2 animals survived). Controlused saline only as an active (n=4 animals).

Topical Application:

The control or Revance's botulinum formulation was applied to the hindlimb using a pipet and massaged into the skin wearing nitrile gloves.DYSPORT® and backbones were stored at 4° C. Animals were incubated in acontrolled heat environment to prevent hypothermia. Muscle contractionforce was measured at 2-3.5 hours post topical treatment.

Muscle Contraction Force Generation:

The limb was immobilized by securing it to a wooden table using K-wiresthrough the femur and the tibia to prevent motion. The gastrocnemius wasleft in situ. A wire suture was tied around the distal end of theAchilles tendon. The tendon was then transected distal to the suture,and the suture was attached to a force transducer (model FT03, Grass,West Warwick, R.I.), which in turn was connected to a force transduceramplifier (model 13-G4615-50, Gould, Cleveland, Ohio). The sciatic nervefrom the DYSPORT® treated side was stimulated directly (SD9 stimulator,Grass, West Warwick, R.I.) with increasing voltage until the maximumisometric single-twitch force was obtained. The frequency of stimulationthen was increased until maximum tetanic force was generated. Twitch isgenerated by stimulation of one motor unit, and tetanus is generated byapplying summation of all motor units by supermaximal stimulation. Thesame procedure was repeated on the control limbs. Responses wererecorded with a calibrated recording oscillation (RS 3800, Gould,Cleveland, Ohio) linked to the force transducer. [Ma J, Elsaidi G A,Smith T L, et al. Time course of recovery of juvenile skeletal muscleafter botulinum toxin A injection. Am. J. Phys. Med. Rehabil. 2004;83(10):774-780].

Results

Normal values of muscle force generation in a C57BL/6 mice has a meansingle twitch force of 60±15 grams and a mean tetanus force of 240±30grams in a previous study with injection of botulinum toxin A. In thispilot preclinical study, comparable mean single twitch force of 54±2grams and mean tetanus force of 241±20 grams were found.

When muscle force generation of topical DYSPORT® with Kn21Pr wasevaluated, it showed no response resulting in approximately 100%decrease in single twitch and tetanus response in animal treated withsingle-time administration of topical Revance's botulinum formulationwith Kn21Pr versus the controls on the recordings whereas muscle forcegeneration showed approximately 58% decrease in single twitch andapproximately 61% decrease in tetanus in animal treated with single-timeadministration of topical Revance's botulinum formulation with KnTversus the controls for the lower limit. Tables 10 and 11 show the meanand standard error for the single twitch test and the tetanus testrespectively.

TABLE 10 Muscle force generation - Single twitch test. Mean and standarderrors for treatment group versus control are presented. Animal groupMean Std. Error treatment 19 10.97 control  45* 0.00 *lower limit

TABLE 11 Muscle force generation - Tetanus test. Mean and standarderrors for treatment group versus control are presented. Animal groupMean Std. Error treatment 81 46.765 control 210* 0.00 *lower limitConclusion:

This study serves to demonstrate that topical application of DYSPORT® at25 units per mouse limb can effectively decrease motor force generationand shows evidence of therapeutic benefits.

Example 9 Evaluating Muscle Force Generation after Topical Applicationof Revance's Botulinum Formulation

Purpose:

To evaluate the effects of neuromuscular blockade after topicalapplication of botulinum toxin type A by muscle contraction forcegeneration in a murine model.

Methods:

Study Design:

Male CD1 mice (Charles River, Wilmington, Mass.) weighing 27-33 g wereused. Mice were housed in groups of 5 and allowed ad libitum access tofood and water before treatment. Animals were anesthetized using 1.5%isoflurane mixed with oxygen and remained anesthetized for the durationof the study. A dose site of each mouse's hind limb was carefully shavedwith an Andis Edjer II cordless rechargeable trimmer (Andis, Sturtevant,Wis.). Botox was applied topically at a dosage of 10 units per mouselimb. Untreated normals, as well as those treated with base formulations(no toxin) applied topically at an equivalent volume served as controls.Muscle contraction force was measured at 2-3.5 hours post topicaltreatment.

Test Compound Preparation:

The BOTOX® reconstituting solution of sterile 0.9% sodium chloride(Abbott Laboratories, North Chicago, Ill.) with 5% EtOH and 5%polyaspartate solution was prepared. Backbones were prepared at 1milligram/milliliter concentration with 0.9% sodium chloride. 100 unitsof BOTOX® (Allergan, Irvine, Calif.) was reconstituted with 1.0milliliters of reconstituting solution using sterile 3 ml latex freesyringe with 18_(G)1½ (Becton Dickinson & Co., Franklin Lakes, N.J.).The reconstituted BOTOX® was carefully mixed by inversion eight times.The “Revance BOTOX® solution” was prepared with 10 units of BOTOX® andbackbone (i.e. 100 microliters of BOTOX® was added to 50 microliters ofproprietary short peptidyl backbone based on prior experiments above) ina microcentrifuge tube and sat at room temperature for 5 minutes for thecomplexes to form (n=2 animals survived). Control used saline only as anactive (n=4 animals).

Procedures:

The control or “Revance BOTOX® solution” was applied to the hind limbusing a pipet and massaged into the skin wearing nitrile gloves. BOTOX®and backbones were stored at 4° C. Animals were incubated in acontrolled heat environment to prevent hypothermia. Muscle contractionforce was measured at 2-3.5 hours post topical treatment.

Muscle Contraction Force Generation:

The limb was immobilized by securing it to a wooden table using K-wiresthrough the femur and the tibia to prevent motion. The gastrocnemius wasleft in situ. A wire suture was tied around the distal end of theAchilles tendon. The tendon was then transected distal to the suture,and the suture was attached to a force transducer (model FT03, Grass,West Warwick, R.I.), which in turn was connected to a force transduceramplifier (model 13-G4615-50, Gould, Cleveland, Ohio). The sciatic nervefrom the BOTOX® treated side was stimulated directly (SD9 stimulator,Grass, West Warwick, R.I.) with increasing voltage until the maximumisometric single-twitch force was obtained. The frequency of stimulationthen was increased until maximum tetanic force was generated. Twitch isgenerated by stimulation of one motor unit, and tetanus is generated byapplying summation of all motor units by supermaximal stimulation. Thesame procedure was repeated on the control limbs. Responses wererecorded with a calibrated recording oscillation (RS 3800, Gould,Cleveland, Ohio) linked to the force transducer. [Ma J, Elsaidi G A,Smith T L, et al. Time course of recovery of juvenile skeletal muscleafter botulinum toxin A injection. Am. J. Phys. Med. Rehabil. 2004;83(10):774-780].

Results:

Normal values of muscle force generation in a C57BL/6 mice as performedhere are mean single twitch force of 60±15 grams and mean tetanus forceof 240±30 grams. In this pilot preclinical study, comparable mean singletwitch force of 54±2 grams and mean tetanus force of 241±20 grams werefound.

When muscle force generation of topical Botox® with Kn21Pr wasevaluated, it showed no response, resulting in approximately 100%decrease in single twitch and tetanus response in animal treated withsingle-time administration of topical “Revance Botox® solution” withKn21Pr versus the controls on the recordings (FIGS. 10(a) and 10(b))whereas muscle force generation showed approximately 90% decrease insingle twitch and 100% decrease (no response) in tetanus in animaltreated with single-time administration of topical “Revance Botox®solution” with KNP versus the controls. Table 12 shows the mean andstandard error for the single twitch test and the tetanus testrespectively. Table 13 shows the summary of mean muscle force generationand percentage of decrease for single twitch and tetanus.

TABLE 12 Muscle force generation - Tetanus test. Mean and standarderrors for treatment group versus control are presented. Animal groupMean Std. Error treatment 4.667 4.667 control 45 0 *lower limit

TABLE 13 Summary of muscle force generation studies from Examples 8 and9. Mean values and % of decrease for treatment group versus control arepresented. Muscle Force Mean Mean % Treatment Carrier Generation Results(g) decrease BOTOX ® Kn21T(2)/Kn21pr(1) single twitch  5 90% tetanus  0100% DYSPORT ® Kn21T/Kn21pr single twitch 19 58% tetanus 81 61% ControlN/A single twitch  45* 0% tetanus 210* 0% *lower limitConclusion:

This study serves to demonstrate that topical application of bothDYSPORT® at 25 units per mouse limb and BOTOX® at 10 units per mouselimb can effectively decrease motor force generation and shows evidenceof therapeutic benefits.

Example 10 In Vitro Carrier-Mediated Transdermal Delivery of BotulinumToxin Type A Across Porcine Skin Pt. 1 Flow-Through Analysis

Purpose:

To evaluate the efficiency of our carrier in delivering Botulinum ToxinType A (BoNTA) across the skin barrier.

Methods:

Biotinylating Toxin:

10 micrograms of Botulinum Toxin Type A (List Biological Laboratories,Campbell, Calif.) was resuspended in 100 microliters of PBS, pH 7.4.Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill.) was added at a 20-foldmolar excess, and the reaction volume was brought to 1 milliliter.Reaction was incubated at room temperature for 1 hour, and dialyzedagainst PBS overnight using a 10 K MWCO Slide-A-Lyzer Dialysis Casette(Pierce, Campbell, Calif.).

Harvesting Skin:

Intact skin was harvested from male and female pig abdomen (Lychron LLC,Mountain View, Calif.). During transport, skin was immersed in an icebath containing PBS, 10 unit/milliliters Penicillin, and 10micrograms/milliliters Streptomycin. Epidermis and dermis were isolatedusing a Dermatome (Padgett Instruments, Plainsboro, N.J.) set at athickness of 0.8 mm. Skin was snap-frozen in liquid nitrogen and storedat −80° C. until use.

Testing:

All experimental conditions were tested in triplicate. For each sample,appropriate amounts of carrier and toxin were added (Table 14) and thevolume was brought to 200 microliters with PBS.

TABLE 14 Description of test sample composition Toxin (μg) 0.05 0.050.05 0.1 0.1 0.1 1 1 1 Carrier K30T K30T K30T K30T K30T K30T K30T K30None Carrier 0.025 0.05 0.1 0.05 0.1 0.2 1 1 — (μg)

Skin was thawed in a 37° C. water bath immediately before use, sectionedinto 1.5 cm×1.5 cm squares, and secured inside the modified FranzChamber apparatus (PermeGear, Bethlehem, Pa.). A Haake DC10 circulatingwater bath (Thermo Electron, Karlsruhe, Germany) was set at 37° C.Samples were applied to the skin surface, and the flow-through rate wasset at 8.02 microliters/minute using an IPC Ismatec peristaltic pump(Idex, Wertheim-Mondfeld, Germany). Flow-through fractions werecollected using a Retriever IV fraction collector (Teledyne Isco,Lincoln, Nebr.) pooled at hours 0-1, 1-2, 2-3, 3-4, 4-6, 6-8, 8-12, and12-20 using an ATM10 Indexing Controller (Permegear, Bethlehem, Pa.).See FIG. 12 for apparatus setup.

Sample Analysis:

Serial dilutions of biotinylated toxin solution were performed forstandard curve. A plate was coated with 200 microliters flow-throughsamples and standards and incubated at room temperature for 2 hours. Theplate was then washed 3 times with 0.1% TWEEN® 20 in PBS (PBST). 200microliters of blocking buffer/well (20% FBS in PBST) was then added andincubated at room temperature for 2 hours. Blocking buffer was removed.100 microliters of Streptavidin-HRP at 1:1000 in 2% FBS in PBST wasadded to each well, and was incubated at room temperature for 1 hour.The plate was then washed 5 times with PBST. 100 microliters OptEIAsubstrate (BD Biosciences) was added to each well and incubated at roomtemperature for 10 minutes to develop. 50 microliters 1N H₂SO₄ was addedto quench the reaction, and absorption was measured at 450 nm.

Results:

Revance carrier showed an increase in toxin delivery (see FIG. 13).Optimizing the toxin concentration and carrier:toxin mass ratio yieldeda statistically significant (P<0.05) increase in toxin delivery versuscontrols.

Example 11 In Vitro Carrier-Mediated Transdermal Delivery of BotulinumToxin Type A Across Porcine Skin Pt. 2 Flow-Through Analysis

Purpose:

To evaluate the efficiency of Revance carriers in delivering BotulinumToxin Type A and Calf Intestinal Phosphatase (CIP) across the skinbarrier in a porcine skin model using modified Franz chambers.

Methods:

Biotinylating Toxin:

10 micrograms of Botulinum Toxin Type A (List Biological Laboratories,Campbell, Calif. and Sigma-Aldrich) was resuspended in 100 microlitersof PBS, pH 7.4. Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill.) was addedat a 20-fold molar excess, and the reaction volume was brought to 1milliliter with PBS. The pH was check to ensure a pH range of 7-9 forcoupling. Reaction was incubated at room temperature for 1 hour, anddialyzed against PBS overnight using a 10 K MWCO Slide-A-Lyzer DialysisCasette (Pierce, Campbell, Calif.).

Harvesting Skin:

Intact skin was harvested from male and female pig abdomens andshoulders (Lychron LLC, Mountain View, Calif.). During transport, skinwas immersed in an ice bath containing PBS, 10 unit/millilitersPenicillin, and 10 micrograms/milliliters Streptomycin. Skin grafts(full thickness including epidermis and dermis) were isolated using aDermatome (Padgett Instruments, Plainsboro, N.J.) set at a thickness of0.8 mm. Skin grafts were snap-frozen in liquid nitrogen and stored at−80° C. until use.

Testing:

Experimental conditions were tested in triplicate. There were threepayload groups and six different carriers. For each sample, appropriateamounts of carrier and toxin were added (Table 15). The payload andcarriers were prepared in following concentrations: Kn8 carriers (Kn8,Kn8R and Kn8T) were prepared at 10 milligrams/milliliter; Kn21T carrierwas prepared at 1 milligram/milliliter; K30T and Kn21pR and BotulinumToxin Type A (List Labs and Sigma toxins) were prepared at 0.1milligram/milliliter; CIP was prepared at 100 units/milliliter.

TABLE 15 Description of test sample composition Payload amount CarrierPBS Sample Payload (uL) Carrier amount (uL) (uL) 1 CIP 100 Kn8 100 0 2CIP 100 Kn8R 100 0 3 CIP 100 Kn8T 100 0 4 CIP 100 Kn21T 2.86 97.14 5List pure neurotoxin 100 K30T 10 90 6 List pure neurotoxin 100 Kn21pR 1090 7 Sigma 100 K30T 10 90 neurotoxin + lactose 8 Sigma 100 Kn21pR 10 90neurotoxin + lactose

Skin was thawed in a 37° C. water bath immediately before use, sectionedinto 1.5 cm×1.5 cm squares, and secured inside the modified FranzChamber apparatus (PermeGear, Bethlehem, Pa.). A Haake DC10 circulatingwater bath (Thermo Electron, Karlsruhe, Germany) was set at 37° C.Samples were applied to the skin surface, and the flow-through rate wasset at 8.02 microliters/minute using an IPC Ismatec peristaltic pump(Idex, Wertheim-Mondfeld, Germany). Flow-through fractions werecollected using a Retriever IV fraction collector (Teledyne Isco,Lincoln, Nebr.) pooled at hours 0-1, 1-2, 2-3, 3-4, 4-6, 6-8, 8-12, and12-20 using an ATM10 Indexing Controller (Permegear, Bethlehem, Pa.).

Sample Analysis:

Performed serial dilutions of biotinylated toxin solution were done forstandard curve. Plate was coated with 200 microliters flow-throughsamples and standards and incubated at room temperature for 2 hours.Plate was then washed 3 times with 0.1% TWEEN® 20 in PBS (PBST). 200microliters of blocking buffer/well (20% FBS in PBST) was then added andincubated at room temperature for 2 hours. Blocking buffer was removed.100 microliters of Streptavidin-HRP at 1:1000 in 2% FBS in PBST wasadded to each well, and was incubated at room temperature for 1 hour.The plate was then washed 5 times with PBST. 100 microliters OptEIAsubstrate (BD Biosciences) was added to each well and incubated at roomtemperature for 10 minutes to develop. 50 microliters 1N H₂SO₄ was addedto quench the reaction, and absorption was measured at 450 nm.

CIP:

Similar procedure as used for the CIP. CIP was chosen as an alternativebecause is very similar to botulinum toxin. It is a globular proteinwith similar molecular weight of 160 kD. It is less expensive and CIPhas high specific activity.

Results:

TABLE 16 Summary of efficiency. Mean efficiency and standard errors areshown in percentages. Efficiency Std. Error Payload Carrier (%) (%) CIPKn8 0.19 n/a CIP Kn8R 0.26 n/a CIP Kn8T 0.34 n/a CIP Kn21T 0.66 0.18List pure neurotoxin K30T 9.17 1.36 List pure neurotoxin Kn21pR 3.560.41 Sigma neurotoxin + lactose K30T 5.44 0.19 Sigma neurotoxin +lactose Kn21pR 4.61 0.59

As shown in Table 16, different carriers impact depth and tropism fordifferent complexes. Shorter backbones (Kn8 series) stay moresuperficial so flow through less readily. TAT penetrates deeper thanoligoarginine for a given backbone length. Less charged species like CIPdo not form as effective particles so do not penetrate as deeply.Complex components such as lactose can shift carrier preferences (orrequire strategies to form stable particles).

Example 12 In Vitro Carrier-Mediated Transdermal Delivery of BotulinumToxin Type A Across Porcine Skin Pt. 3 Flow-Through Analysis

Purpose:

To evaluate the efficiency of Revance carrier in delivering BotulinumToxin Type A across the skin barrier in a porcine skin model usingmodified Franz chambers.

Methods:

Harvesting Skin:

Intact skin was harvested from female pig shoulder and abdomen (LychronLLC, Mountain View, Calif.). During transport, skin was immersed in PBS,containing 10 unit/ml Penicillin, and 10 mg/ml Streptomycin and kept iton ice. Skin grafts (full thickness including epidermis and dermis) wereisolated using a Dermatome (Padgett Instruments, Plainsboro, N.J.) setat a thickness of 0.8 mm and were snap-frozen in liquid nitrogen andstored at −80° C. until use.

Biotinylating Toxin:

10 μg of Botulinum Toxin Type A (List Biological Laboratories, Campbell,Calif.) was resuspended in 100 μl of PBS, pH 7.4. Sulfo-NHS-LC-Biotin(Pierce, Rockford, Ill.) was added at a 100-fold molar excess, and thereaction volume was brought to 1 ml with PBS. The pH was check to ensurea pH range of 7-9 for coupling. Reaction was incubated at roomtemperature for 2 hours and dialyzed against PBS overnight using a 10 KMWCO Slide-A-Lyzer Dialysis Casette (Pierce, Campbell, Calif.). Nextday, biotinylated toxin is aliquoted (100 μl/tube) and stored at 4° C.

Preparation of Skin for Franz Chamber:

Skin was thawed in a 37° C. water bath immediately before use, sectionedinto 1.5 cm×1.5 cm squares, and secured inside the modified FranzChamber apparatus (PermeGear, Bethlehem, Pa.). A Haake DC10 circulatingwater bath (Thermo Electron, Karlsruhe, Germany) was set at 37° C.Samples were applied to the skin surface, and the flow-through rate wasset at 8.02 μl/minute using an IPC Ismatec peristaltic pump (Idex,Wertheim-Mondfeld, Germany). Flow-through fractions were collected usinga Retriever IV fraction collector (Teledyne Isco, Lincoln, Nebr.) pooledat hours 0-1, 1-2, 2-3, 3-4 using an ATM10 Indexing Controller(Permegear, Bethlehem, Pa.).

Formulations:

For each sample, appropriate amounts of carrier and toxin were added.All experimental conditions were tested in quadruplicates. The treatmentgroup was biotinylated toxin with Revance carriers and the control groupwas biotinylated toxin only. Carrier and Botulinum Toxin Type A wereprepared in different mass ratio in order to optimize toxinconcentration and toxin:carrier mass ratios.

ELISA (Protein Quantification Assay):

Serial dilutions (1:3 and 1:2) of biotinylated toxin were done forstandard curve. Plate was coated with 200 μl flow-through samples andstandards and incubated at room temperature for 2 hours. After 2 hours,samples and standards were discarded, the plate was blocked with 300 μlof superblock blocking buffer to each well for 1 minute at roomtemperature and repeated two times. Blocking buffer was removed and 100μl of Streptavidin-HRP at 1:1000 in PBST with 2% FBS was added to eachwell, and was incubated at room temperature for 1 hour. The plate wasthen washed with 300 μl (per well) PBST (PBS with 0.1% TWEEN® 20) for 5minutes at room temperature and repeated 2 times. After washing, 100 μlof OptEIA substrate (BD Biosciences) was added to each well andincubated at room temperature for 10 minutes to develop. Then 50 μl 1NH₂SO₄ was added to quench the reaction, and absorption was measured at450 nm.

Results:

Revance carrier showed an increase in toxin delivery compared to thecontrols (see FIGS. 14a-f ).

Example 13 In Vitro Carrier-Mediated Transdermal Delivery of BotulinumToxin Type A Across Porcine Skin Skin Analysis

Purpose:

To evaluate the efficiency of Revance carrier in delivering BotulinumToxin Type A across the skin barrier in a porcine skin model usingmodified Franz chambers.

Methods:

Biotinylating Toxin:

10 micrograms of Botulinum Toxin Type A (List Biological Laboratories,Campbell, Calif.) was resuspended in 100 microliters of PBS, pH 7.4.Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill.) was added at a 20-foldmolar excess, and the reaction volume was brought to 1 milliliter withPBS. The pH was check to ensure a pH range of 7-9 for coupling. Reactionwas incubated at room temperature for 1 hour, and dialyzed against PBSovernight using a 10 K MWCO Slide-A-Lyzer Dialysis Casette (Pierce,Campbell, Calif.).

Harvesting Skin:

Intact skin was harvested from female pig shoulder (Lychron LLC,Mountain View, Calif.). During transport, skin was immersed in an icebath containing PBS, 10 unit/milliliters Penicillin, and 10micrograms/milliliters Streptomycin. Skin grafts (full thicknessincluding epidermis and dermis) were isolated using a Dermatome (PadgettInstruments, Plainsboro, N.J.) set at a thickness of 0.8 mm. Skin graftswere snap-frozen in liquid nitrogen and stored at −80° C. until use.

Testing:

All experimental conditions were tested in triplicate. There were twotest groups. The treatment group was biotinylated toxin with RevanceK30Ts carrier and the control group was biotinylated toxin only. Foreach sample, appropriate amounts of carrier and toxin were added (Table17). Carrier and Botulinum Toxin Type A were prepared at 0.1milligram/milliliter concentration.

TABLE 17 Description of test sample composition Payload Carrier SamplePayload amount (μL) Carrier amount (μL) PBS (μL) 1 List toxin 100 K30Ts10 90 2 List toxin 100 K30Ts 10 90 3 List toxin 100 K30Ts 10 90 4 Listtoxin 100 n/a N/A 90 5 List toxin 100 n/a N/A 90 6 List toxin 100 n/aN/A 90

Skin was thawed in a 37° C. water bath immediately before use, sectionedinto 1.5 cm×1.5 cm squares, and secured inside the modified FranzChamber apparatus (PermeGear, Bethlehem, Pa.). A Haake DC10 circulatingwater bath (Thermo Electron, Karlsruhe, Germany) was set at 37° C.Samples were applied to the skin surface, and the flow-through rate wasset at 8.02 microliters/minute using an IPC Ismatec peristaltic pump(Idex, Wertheim-Mondfeld, Germany). Flow-through fractions werecollected using a Retriever IV fraction collector (Teledyne Isco,Lincoln, Nebr.) pooled at hours 0-1, 1-2, 2-3, 3-4, 4-6, 6-8, 8-12, and12-20 using an ATM10 Indexing Controller (Permegear, Bethlehem, Pa.).

Streptavidin Staining:

The snap frozen skin was sectioned and hydrated with 0.1% TWEEN® 20 inPBS (PBST). The sections were blocked with BLOTTO® blocking buffer for 2hours at room temperature and then rinsed with PBST for 5 minutesintervals three times. Streptavidin-HRP at 1:1000 in 2% FBS in PBST wasadded and the sections were incubated at room temperature for 30 minutesand then rinsed with PBST for 5 minutes intervals three times. OptEIAsubstrate (BD Biosciences) was added and the sections were incubated atroom temperature for 3 minutes to develop. The sections were washed withPBST and covered with a coverslip.

Results:

Photographs:

Stained sections were photographed with a Retina 1300B camera (Imaging,Burnaby, BC, Canada) on a Nikon E600 microscope with 4× magnificationplan-apochromat objective lenses (FIG. 15).

Example 14 Effectiveness of Botulinum Toxin Type A in Causing MuscleParalysis

Objective

To study the effectiveness of botulinum toxin type A in causing muscleparalysis in mice after topical application of toxin in Revance'sbotulinum formulation by muscle force generation test.

Materials and Methods:

Botulinum Toxin:

NEURONOX® (Medy-Tox, Inc., Korea) Lot#NNX0502, 100 U/vial;

0.9% NaCl (no preservatives, Abbott Laboratories, North Chicago, Ill.)

Polylysine Peptide (Carrier):

K15T2

Test groups - 10 U toxin/mouse, n = 4 K15T2 carrier No carrierNEURONOX ® (NNX) x xPolylysine Peptide/Toxin Complexes Preparation:Target carrier: albumin/toxin mass ratio=1.1:1Neuronox Preparation: (0.5 mg Albumin/100 U Toxin)

A 1.1 mg/ml stock solution of K15T2 carrier with 0.9% NaCl in amicrocentrifuge tube was made and mixed by vortex. 100 U (1 vial) of NNXwas reconstituted with 0.5 ml of 0.9% NaCl by slowly injecting 0.9% NaClinto the side wall of a vial and mixed by gentle inversion. (Theconcentration was 100 U toxin/0.5 ml). 0.5 ml carrier stock solution wasslowly injected from 0.5 ml reconstituted toxin and gently inverted thecarrier/toxin vial 10 times, sat at room temperature for 5 minutes withthe vial standing upright and then gently inverted again before dosing.(The concentration was 100 U toxin/1.0 ml). 100 μl of the mixture wastaken out with 1 ml syringe and a needle. The needle was removed beforeapplication. (Final concentration is 10 U toxin/100 μl).

Study Design:

Male CD1 mice (Charles River, Wilmington, Mass.) weighing 22-26 g wereused for treatment and 28-31 g for control. Mice were housed in groupsof 5 and allowed ad libitum access to food and water before treatment.Animals were anesthetized using 1.5% isoflurane mixed with oxygen andremained anesthetized for the duration of the treatment with a dwelltime of 30 minutes. A dose site of each mouse's hind limb was carefullyshaved with an Andis Edjer II cordless rechargeable trimmer (Andis,Sturtevant, Wis.). NNX was applied topically at a dosage of 10 units permouse limb. Toxin without peptidyl carrier applied topically at anequivalent volume served as controls. Muscle contraction force wasmeasured at day 4 post topical treatment.

Topical Application:

Animals were anesthetized with isoflurane mixed with oxygen. 100 μl (10units) of the carrier/toxin mixture was slowly applied by using 1 mlsyringe without a needle to spread to a randomly selected hind leg ofthe mouse (shaved). The other leg is treated with the control mixture.The mixture was massaged onto the hind leg while wearing nitrile gloves,incubated for 30 minutes while the mouse was under anesthesia, then thedosing site was rinsed with water and wiped with a paper towel to cleanoff the residue toxin at dose site. After topical application andcleansing, mice were returned to its cage for housing and recovered fromanesthesia in heat controlled environment. Animals were observed ondaily basis for behavioral signs of systemic toxicity such as reducedrespiratory rate, labored breathing, ptosis and mydriasis or muscleparalysis. Muscle force generation was measured at day 4 post treatment.

Muscle Contraction Force Generation:

The limb was immobilized by securing it to a wooden table using K-wiresthrough the femur and the tibia to prevent motion. The gastrocnemius wasleft in situ. A wire suture was tied around the distal end of theAchilles tendon. The tendon was then transected distal to the suture,and the suture was attached to a force transducer (model FT03, Grass,West Warwick, R.I.), which in turn was connected to a force transduceramplifier (model 13-G4615-50, Gould, Cleveland, Ohio). The sciatic nervefrom the NNX treated side was stimulated directly (SD9 stimulator,Grass, West Warwick, R.I.). The frequency of stimulation was increaseduntil maximum tetanic force was generated. Tetanus is generated byapplying summation of all motor units by supermaximal stimulation. Thesame procedure was repeated on the control limbs. Responses wererecorded with a calibrated recording oscillation (RS 3800, Gould,Cleveland, Ohio) linked to the force transducer. [Ma J, Elsaidi G A,Smith T L, et al. Time course of recovery of juvenile skeletal muscleafter botulinum toxin A injection. Am. J. Phys. Med. Rehabil. 2004; 83(10):774-780].

Results:

When muscle force generation of topical NNX with peptidyl carrier systemwas evaluated, it showed reduction in muscle force resulting inapproximately 55% decrease in tetanus response in animal treated withsingle-time topical administration, which had a mean of 128±11 grams forNNX with K15T2 (treatment group) and 286±16 grams for NNX withoutcarrier (control group) (Table 18). Summary of stimulation frequencywhich includes average percentage of muscle force reduction, mean andstandard error for each stimulation frequency are presented in Table 19.FIG. 16 shows the percentage of muscle for reduction for NNX with K15T2(treatment group), NNX without carrier (control group), and NNXinjection.

TABLE 18 Muscle force generation - Tetanus test. Mean and standarderrors for treatment group versus control are presented. Animal groupMean Std. Error treatment 128 11 control 286 16

TABLE 19 Summary of stimulation frequency Percentage of muscle forcereduction, mean and standard errors are presented. max tetanus tetanustetanus tetanus tetanus tetanus force 25 Hz 40 Hz 60 Hz 100 Hz 150 Hz200 Hz Stimulation frequency (Hz) grams grams grams grams grams gramsgrams NNX + Carrier Group (10 U/mouse) % muscle force reduction vs.internal control std. error 14% 15% 5% 6% 14% 18% average 49% 34% 20%30% 32% 39% Mean 24 30 70 116 128 125 109 Std. error 8 8 16 7 11 25 33NNX Alone - NO Carrier Group (10U/mouse) % muscle force reduction vs.internal control std. error 14% 14% 19% 12% 9% 4% 2% average 3% 21% 3%1% −2% −2% −2% Mean 63 76 155 233 284 283 267 Std. error 9 14 30 28 1610 6Conclusion:

This study demonstrated that botulinum toxin A in Revance's botulinumformulation in proprietary carrier system was effective in causingmuscle paralysis in mice after single-time topical application.

Example 15 Human Pilot Study Topical Botulinum Toxin Type A to ReduceForehead Wrinkles without Functional Impairment of the Expression

Topical Botulinum Toxin Preparation and Application:

Subject 1:

First treatment—4 μg of K15T2 Revance Carrier was dissolved in 2.0 ml of15% poloxamer (in 0.9% sodium chloride and no EtOH) and mixed byinversion. 1.0 ml of poloxamer-Carrier mixture was used to reconstitute400 U of BOTOX® (Allergan, Irvine, Calif.) in serial steps and mixed byinversion. The final concentration of Carrier to toxin ratio was 0.5 mgCarrier/100 U toxin. The topical toxin solution sat at room temperaturefor 5 minutes. The total volume of 1.0 ml was applied to forehead usinga syringe. After a 30 minute incubation, the treated forehead area waswashed with five wet paper towels, where each paper towel was used towipe the forehead once in one direction only. Photographs weredocumented at pre-treatment (baseline) and post-treatment.

Subject 2:

First treatment—10 μg of botulinum toxin type A (List BiologicalLaboratories, Inc., Campbell, Calif., product #130A) was reconstitutedin 1.0 ml of 0.9% sodium chloride and the further diluted to a finalconcentration of 5 ng/100 μl. Reconstituted toxin was mixed byinversion. Revance Carrier was prepared at a concentration 1 ng/20 μl. 1ng of Carrier was mixed in 900 μl 15% poloxamer in 0.9% NaCl (no EtOH).And then, 100 ml of toxin was added to 920 μl of poloxamer to make a1:10:toxin:diluent ratio. The total volume of 1.02 ml was applied toforehead using a syringe. After 30 minute incubation, treated foreheadarea was washed with five wet paper towels in the same manner assubject 1. Photographs were documented at pre-treatment (baseline) andpost-treatment.

Results:

FIG. 17 shows photographs taken before and after the treatment. Thephotographs show reduced forehead wrinkles after topical botulinum toxintype A. Human subject 1 (top row) and subject 2 (bottom row) photographspre-treatment (baseline) and post-treatment.

Example 16 Hyperhidrosis Study Revance Topical Botulinum Formulation toTreat Excessive Sweating in Human Subjects

Purpose:

To determine feasibility of sweat inhibition by topical application ofRevance's botulinum formulation.

Preparation:

1 mg/ml of each KNR and P6R-B backbones were prepared in deionized H₂Oin separate tubes, vortexed to mix well. BOTOX®—100 units (Allergan,Irvine, Calif.) was reconstituted with either 1.0 ml of deionized H₂O(subject 1) or 0.5 ml of 0.9% NaCl (subject 2 & 3) and mixed byinversion. Stock solution of the treatments was prepared by adding theingredients shown in the following table to a 1.7 ml microcentrifugetube, vortexing for 90 seconds, and incubating at room temperature for 5minutes for allow the complexes to form. The conical tubes were labeledas R for right and L for left. 1.0 ml of stock solution was transferredto assigned conical tubes, 1:5 dilution was prepared by adding 4.0 ml ofCetaphil (Galderma, Fort Worth, Tex.) and mixed well using a metalspatula.

Ratio Subject Botox (U) Carrier (toxin:diluent) Total volume 1 20 KNR5:8 1.3 ml 2 50 KNR 1:1 1.5 ml

Gravimetric Assessment:

Each step of the procedure was performed while wearing nitrile (orpowder free) gloves. Three layers of filter paper were prepared andplaced in a conical tube using forceps making sure that filter paperswere near the screwed top. The filter papers were preweighed in thetubes by placing a tube on its screwed top in the center of the balancepan. Three sets of filter papers were preweighed per subject. The filterpapers were placed on each axilla simultaneously. The subjects sat inresting position with their arms by their sides and hands folded infront of the body. Subjects remained still for the collection period ina heated room. After each 10 minute sweat collection period, the filterpapers were removed and returned to the their tubes. The filter paperswere placed in its original position, unfolded and near the screwed topusing forceps using care not to rip the filter papers. Baseline sweatmeasurements were collected at 10 minutes intervals three times. Thefilter papers were reweighed by placing a tube on its screwed top in thecenter of the balance pan and the weight was recorded.

Drug Application Procedure:

While wearing nitrile gloves, 5.0 ml (subject 1) and 1.5 ml (for subject2) of test article or control was topically applied to axilla using ametal spatula (for subject 1) or rolling applicator (in up and downmotion, then circular motion for subject 2). In case of subject 2, eachaxilla was coated with approximately 4 ml of Cetaphil using a smallspatula and incubated for 1 hour at room temperature (72-77° F.) toallow the treatment to be absorbed. Axillae were wiped and cleansed withcleansing cloth (Johnson & Johnson).

Observations:

Hair on axillae was intact. Waxing was not done before treatmentapplication. Subjects stated that there was no pain or discomfort. Bothsides of axilla had tingling sensation. There were no signs ofirritation and no change in skin pigment.

Results

Gravimetric Analysis (Human Subject 1):

Over the first 7 days post topical application of 20 U botulinum toxin(BOTOX®) each axilla with or without the Revance peptidyl carrier forhuman subject #1 in feasibility trial. Sweat produced (mg) per 10minutes under standard conditions (p=0.0043)

botulinum alone Revance's botulinum (control) formulation %cross-section 20.5 13.4

Gravimetric Analysis (Human Subject 2):

Over the first 7 days post topical application of 50 U botulinum toxin(BOTOX®) each axilla with or without the Revance peptidyl carrier forhuman subject #2 in feasibility trial. Sweat produced (mg) per 10minutes under standard conditions (p=0.0117). FIG. 18 shows a photographof subject 2 after application of a botulinum toxin formulationaccording to the invention in one axilla, and the application of acontrol formulation in the other.

botulinum alone Revance's botulinum (control) formulation %cross-section 201 157

What is claimed is:
 1. A method of treating wrinkles, comprising:applying to an area of skin of a patient in need of treating wrinkles aformulation comprising: a botulinum toxin, a carrier comprising apositively charged polymeric backbone with positively charged efficiencygroups covalently attached thereto, a poloxamer; and a dermatologicallyor pharmaceutically acceptable excipient, diluent, or medium; whereinthe botulinum toxin is non-covalently associated with the positivelycharged backbone; and wherein the carrier comprising the positivelycharged backbone is the sole necessary agent for non-covalentlyassociating with the botulinum toxin, and, optionally, applying anocclusion agent afterwards.
 2. The method according to claim 1, whereinthe area of the skin is selected from the group consisting of the face,forehead, neck, hands and feet.
 3. The method according to claim 1,wherein the positively charged polymeric backbone has a molecular weightof less than 21,000.
 4. The method according to claim 1, wherein thepositively charged polymeric backbone is polylysine or polyethyleneimine(PEI).
 5. The method according to claim 4, wherein the positivelycharged efficiency groups are either protected oligoarginine or TATdomains.
 6. The method according to claim 5, wherein the formulationfurther comprises hydroxypropylcellulose (HPC) or polyethylene glycol(PEG).
 7. The method according to claim 5, wherein the positivelycharged efficiency groups include an the amino acid sequence selectedfrom the group consisting of (gly)_(p)-RGRDDRRQRRR-(gly)_(q) (SEQ ID NO:2), (gly)_(p)-YGRKKRRQRRR-(gly)_(q) (SEQ ID NO: 3), and(gly)_(p)-RKKRRQRRR-(gly)_(q) (SEQ ID NO: 4), wherein the subscripts pand q are each independently an integer of from 0 to
 20. 8. The methodaccording to claim 7, wherein the positively charged efficiency groupsinclude the amino acid sequence (gly)_(p)-RGRDDRRQRRR-(gly)_(q) (SEQ IDNO: 2), wherein the subscripts p and q are each independently an integerof from 0 to
 20. 9. The method according to claim 7, wherein thepositively charged efficiency groups include the amino acid sequence(gly)_(p)-YGRKKRRQRRR-(gly)_(q) (SEQ ID NO: 3), wherein the subscripts pand q are each independently an integer of from 0 to
 20. 10. The methodaccording to claim 7, wherein the positively charged efficiency groupsinclude the amino acid sequence (gly)_(p)-RKKRRQRRR-(gly)_(q) (SEQ IDNO: 4), wherein the subscripts p and q are each independently an integerof from 0 to
 20. 11. The method according to claim 7, wherein thesubscripts p and q are each independently an integer of from 0 to
 8. 12.The method according to claim 7, wherein the subscripts p and q are eachindependently an integer of from 2 to
 5. 13. The method according toclaim 7, wherein the positively charged efficiency groups has the aminoacid sequence (gly)_(p)-RGRDDRRQRRR-(gly)_(q) (SEQ ID NO: 2), whereinthe subscripts p and q are each independently an integer of from 2 to 5.14. The method according to claim 7, wherein the positively chargedefficiency groups has the amino acid sequence(gly)_(p)-YGRKKRRQRRR-(gly)_(q) (SEQ ID NO: 3), wherein the subscripts pand q are each independently an integer of from 2 to
 5. 15. The methodaccording to claim 7, wherein the positively charged efficiency groupshas the amino acid sequence (gly)_(p)-RKKRRQRRR-(gly)_(q) (SEQ ID NO:4), wherein the subscripts p and q are each independently an integer offrom 2 to
 5. 16. The method according to 1, wherein the botulinum toxinis of a serotype selected from the group consisting of serotypes A, B,C, D, E, F, G and mixtures thereof.
 17. The method according to 16,wherein the botulinum toxin is of serotype A.
 18. The method accordingto claim 7, wherein the positively charged polymeric backbone ispolylysine.
 19. The method according to claim 13, wherein the positivelycharged polymeric backbone is polylysine.
 20. The method according toclaim 14, wherein the positively charged polymeric backbone ispolylysine.
 21. The method according to claim 15, wherein the positivelycharged polymeric backbone is polylysine.
 22. The method according toclaim 18, wherein the botulinum toxin is of serotype A.
 23. The methodaccording to claim 19, wherein the botulinum toxin is of serotype A. 24.The method according to claim 20, wherein the botulinum toxin is ofserotype A.
 25. The method according to claim 21, wherein the botulinumtoxin is of serotype A.
 26. The method according to claim 22, whereinthe botulinum toxin has a molecular weight of 150,000.
 27. The methodaccording to claim 23, wherein the botulinum toxin has a molecularweight of 150,000.
 28. The method according to claim 24, wherein thebotulinum toxin has a molecular weight of 150,000.
 29. The methodaccording to claim 25, wherein the botulinum toxin has a molecularweight of 150,000.
 30. The method according to claim 18, wherein thepolylysine has a molecular weight of about 21,000.
 31. The methodaccording to claim 19 wherein the polylysine has a molecular weight ofabout 21,000.
 32. The method according to claim 20, wherein thepolylysine has a molecular weight of about 21,000.
 33. The methodaccording to claim 21, wherein the polylysine has a molecular weight ofabout 21,000.
 34. The method according to claim 18, wherein the aminoacid sequence is attached to the polylysine via either the C-terminus orthe N-terminus of the amino acid sequence.
 35. The method according toclaim 19, wherein the amino acid sequence is attached to the polylysinevia either the C-terminus or the N-terminus of the amino acid sequence.36. The method according to claim 20, wherein the amino acid sequence isattached to the polylysine via either the C-terminus or the N-terminusof the amino acid sequence.
 37. The method according to claim 21,wherein the amino acid sequence is attached to the polylysine via eitherthe C-terminus or the N-terminus of the amino acid sequence.